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1

Twist solitons in complex macromolecules: From DNA to polyethylene  

Microsoft Academic Search

DNA torsion dynamics is essential in the transcription process; simple models for it have been proposed by several authors, in particular Yakushevich (Y model). These are strongly related to models of DNA separation dynamics such as the one first proposed by Peyrard and Bishop (and developed by Dauxois, Barbi, Cocco and Monasson among others), but support topological solitons. We recently

Mariano Cadoni; Roberto De Leo; Sergio Demelio; Giuseppe Gaeta

2008-01-01

2

Comparative effects of adriamycin and DNA-non-binding analogues on DNA, RNA, and protein synthesis in vitro  

Microsoft Academic Search

Drug-DNA binding is claimed to be the basis by which the antitumor antibiotic adriamycin (doxorubicin) inhibits DNA and RNA synthesis in vitro. However, in preliminary studies the DNA-non-binding adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD 32) showed somewhat greater inhibition of DNA and RNA synthesis than adriamycin under identical conditions. The kinetics of macromolecule synthesis inhibition induced by adriamycin and AD 32, and

Mervyn Israel; John M. Idriss; Yoshihiro Koseki; Vinod K. Khetarpal

1987-01-01

3

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

PubMed Central

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

Hsu, Tracy; Mitragotri, Samir

2011-01-01

4

Theory of twisting and bending of chain macromolecules; analysis of the fluorescence depolarization of DNA  

NASA Astrophysics Data System (ADS)

An elastic model of semiflexible chain macromolecules is developed in order to treat internal rotatory Brownian motion in the DNA helix. Dynamical equations for torsion and bending of the chain are generated, using results from classical elasticity and hydrodynamic theories. The rotational diffusion equation in normal coordinates is derived, and the initial-boundary value problem solved for the conditions of a nanosecond fluorescence depolarization experiment. The resulting time distribution function of the angular orientation of a fluorescent probe, embedded in a chain at thermal equilibrium, is used to compute the emission anisotropy. The predicted decay law is unusual, with exponentials in ~t due to twisting and in ~t1/4 due to bending. Comparison with published data for ethidium-DNA complex reveals that the decay of the anisotropy arises primarily from twisting of the DNA helix, with a small contribution from bending. By fitting theory and experiment, the torsional rigidity C of DNA may be obtained.

Barkley, Mary D.; Zimm, Bruno H.

1979-03-01

5

Diffraction in resonant electron scattering from helical macromolecules: Effects of the DNA backbone  

NASA Astrophysics Data System (ADS)

We recently developed a theoretical framework to treat low-energy electron scattering from helical macromolecules. In this article, we use this framework to extend our previous model of simple base-pair scatterers, organized into the DNA structure, to include the backbone. The internal diffraction pattern due to base pairs is still present, but addition of the backbone screens the base pairs by a factor of 2. More interestingly, the effect of constructive interference on the phosphate groups within the backbone itself is seen to be strong at lower energies. We perform a calculation for electrons incident perpendicular and parallel to the axis of a fragment and find comparable electron patterns on the phosphate groups at the surface of films consisting of vertically or horizontally arranged segments relative to the substrate.

Caron, Laurent; Sanche, Léon

2005-09-01

6

Relating Macromolecular Function and Association: The Structural Basis of Protein–DNA and RNA Recognition  

Microsoft Academic Search

The interaction between proteins and DNA or RNA plays an essential part in the function of biological macromolecules, and\\u000a its physical basis resides in the three-dimensional structure of the interfaces that they form. We analyze the geometric,\\u000a chemical and physical chemical properties of the interfaces that occur in sets of protein–DNA and protein–RNA complexes issued\\u000a from X-ray studies and deposited

Joël Janin; Ranjit P. Bahadur

2008-01-01

7

Adsorption of Macromolecules on Mineral Fibers  

Microsoft Academic Search

Several mineral fibers were shown to adsorb differentially to three classes of biologically significant macromolecules (i.e., DNA, RNA, and protein). The cytotoxicity exerted by the particulates on a normal human fibroblast cell line, with the exception of attapulgite, correlated positively with the degree of macromolecular adsorption exhibited by these substances, namely: short chrysotile > attapulgite = intermediate chrysotile > amosite

Ming J. W. Chang; Laurie B. Joseph; Ralph E. Stephens; Ronald W. Hart

1983-01-01

8

RNA-templated DNA origami structures.  

PubMed

Using the RNA transcript as a template, RNA-templated DNA origami structures were constructed by annealing with designed DNA staple strands. RNA-templated DNA origami structures were folded to form seven-helix bundled rectangular structures and six-helix bundled tubular structures. The chemically modified RNA-DNA hybrid origami structures were prepared by using RNA templates containing modified uracils. PMID:23446278

Endo, Masayuki; Yamamoto, Seigi; Tatsumi, Koichi; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi

2013-04-11

9

DNA-free RNA preparations from mycobacteria  

Microsoft Academic Search

BACKGROUND: To understand mycobacterial pathogenesis analysis of gene expression by quantification of RNA levels becomes increasingly important. However, current preparation methods yield mycobacterial RNA that is contaminated with chromosomal DNA. RESULTS: After sonication of RNA samples from Mycobacterium smegmatis genomic DNA is efficiently removed by DNaseI in contrast to untreated samples. CONCLUSIONS: This procedure eliminates one of the most prevalent

Joachim Stephan; Johannes G Bail; Fritz Titgemeyer; Michael Niederweis

2004-01-01

10

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.

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

2009-01-01

11

A computational framework for mechanical response of macromolecules: application to the salt concentration dependence of DNA bendability.  

PubMed

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

2009-05-01

12

Size-dependent trajectories of DNA macromolecules due to insulative dielectrophoresis in submicrometer-deep fluidic channels  

PubMed Central

In this paper, we demonstrate for the first time that insulative dielectrophoresis can induce size-dependent trajectories of DNA macromolecules. We experimentally use ? (48.5 kbp) and T4GT7 (165.6 kbp) DNA molecules flowing continuously around a sharp corner inside fluidic channels with a depth of 0.4 ?m. Numerical simulation of the electrokinetic force distribution inside the channels is in qualitative agreement with our experimentally observed trajectories. We discuss a possible physical mechanism for the DNA polarization and dielectrophoresis inside confining channels, based on the observed dielectrophoresis responses due to different DNA sizes and various electric fields applied between the inlet and the outlet. The proposed physical mechanism indicates that further extensive investigations, both theoretically and experimentally, would be very useful to better elucidate the forces involved at DNA dielectrophoresis. When applied for size-based sorting of DNA molecules, our sorting method offers two major advantages compared to earlier attempts with insulative dielectrophoresis: Its continuous operation allows for high-throughput analysis, and it only requires electric field strengths as low as ?10 V?cm.

Parikesit, Gea O. F.; Markesteijn, Anton P.; Piciu, Oana M.; Bossche, Andre; Westerweel, Jerry; Young, Ian T.; Garini, Yuval

2008-01-01

13

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

PubMed Central

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.

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

1990-01-01

14

Viral RNA-dependent DNA Polymerase: RNA-dependent DNA Polymerase in Virions of RNA Tumour Viruses  

Microsoft Academic Search

Two independent groups of investigators have found evidence of an enzyme in virions of RNA tumour viruses which synthesizes DNA from an RNA template. This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can

David Baltimore

1970-01-01

15

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

PubMed Central

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

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

2013-01-01

16

In vivo single-cell electroporation for transfer of DNA and macromolecules  

Microsoft Academic Search

Single-cell electroporation allows transfection of plasmid DNA or macrocmolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other

Jennifer E Bestman; Rebecca C Ewald; Shu-Ling Chiu; Hollis T Cline

2006-01-01

17

The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins  

Microsoft Academic Search

The gastrointestinal tract (GIT) of mammals is the main portal of entry for foreign DNA and proteins. We have documented the fate of orally administered DNA or protein in the GIT of the mouse. The gene for the Green Fluorescent Protein (GFP) (4.7 kb) and the genomes of bacteriophage M13 (7.25 kb) and adenovirus type 2 (Ad2; 35.9 kb) were used as test

M. Palka-Santini; B. Schwarz-Herzke; M. Hösel; D. Renz; S. Auerochs; H. Brondke; W. Doerfler

2003-01-01

18

Incorporation of DNA and protein precursors into macromolecules by bacteria at -15 degrees C.  

PubMed

DNA and protein precursors were incorporated into trichloroacetic acid-precipitated material by bacterial cell suspensions during incubation for 50 to 100 days at -15 degrees C. Incorporation did not occur at -70 degrees C and was inhibited by antibiotics. The results demonstrate that bacteria can perform macromolecular synthesis under conditions that mimic entrapment in glacial ice. PMID:12450874

Christner, Brent C

2002-12-01

19

Statistical mechanical treatment of the structural hydration of biological macromolecules: Results for B-DNA  

NASA Astrophysics Data System (ADS)

We constructed an efficient and accurate computational tool based on the potentials-of-mean-force approach for computing the detailed hydrophilic hydration of complex molecular structures in aqueous environments. Using the pair and triplet correlation functions database previously obtained from computer simulations of the simple point charge model of water, we computed the detailed structural organization of water around two B-DNA molecules with sequences d(AATT)3.d(AATT)3 and d(CCGG)3.d(CCGG)3, and canonical structure. [A, T, C, and G denote adenine, thymine, cytosine, and guanine, respectively, and d(...) denotes the deoxyribose in the sugar-phosphate backbone.] The results obtained are in agreement with the experimental observations. A.T base-pair stretches are found to support the marked minor-groove ``spines of hydration'' observed in x-ray crystal structures. The hydrophilic hydration of the minor groove of the molecule d(CCGG)3.d(CCGG)3 exhibits a double ribbon of high water density, which is also in agreement with x-ray crystallography observations of C.G base-pair regions. The major grooves, on the other hand, do not show a comparably strong localization of water molecules. The quantitative results are compared with a computer simulation study of Forester et al. [Mol. Phys. 72, 643 (1991)]. We find good agreement for the hydration of the -NH2 groups, the cylindrically averaged water density distributions, and the overall hydration number. The agreement is less satisfactory for the phosphate groups. However, by refining the treatment of the anionic oxygens on the phosphate groups, almost full quantitative agreement is achieved.

Hummer, Gerhard; Soumpasis, Dikeos Mario

1994-12-01

20

Chimeric RNA\\/DNA oligonucleotide-based gene therapy  

Microsoft Academic Search

Chimeric RNA\\/DNA oligonucleotide-based gene therapy.BackgroundChimeric RNA\\/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models.MethodsSix recent studies of chimeric RNA\\/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA\\/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site

Li-Wen Lai; Yeong-Hau H. Lien

2002-01-01

21

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

Microsoft Academic Search

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

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

2006-01-01

22

The interaction of TFIIIA with specific RNA-DNA heteroduplexes.  

PubMed

We examine the association of transcription factor TFIIIA with RNA-DNA heteroduplexes containing sequences from the Xenopus borealis 5 S rRNA gene. Under conditions where TFIIIA selectively binds to 5 S rRNA or the internal control region of the 5 S rRNA gene, no specific association of TFIIIA with DNA-RNA heteroduplexes containing either strand of 5 S DNA could be detected. We discuss our results with respect to specific models of TFIIIA recognition of the internal control region and of DNA-RNA hybrids by zinc finger proteins. PMID:7559384

Nightingale, K P; Wolffe, A P

1995-09-29

23

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA  

Microsoft Academic Search

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of

Yuri L. Lyubchenko; Alexander A. Gall; Lyuda S. Shlyakhtenko; Rodney E. Harrington; Bertram L. Jacobs; Patrick I. Oden; Stuart M. Lindsay

1992-01-01

24

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

25

Synergistic self-assembly of RNA and DNA molecules  

PubMed Central

DNA has recently been used as a programmable ‘smart’ building block for the assembly of a wide range of nanostructures. It remains difficult, however, to construct DNA assemblies that are also functional. Incorporating RNA is a promising strategy to circumvent this issue as RNA is structurally related to DNA but exhibits rich chemical, structural and functional diversities. However, only a few examples of rationally designed RNA structures have been reported. Herein, we describe a simple, general strategy for the de novo design of nanostructures in which the self-assembly of RNA strands is programmed by DNA strands. To demonstrate the versatility of this approach, we have designed and constructed three different RNA–DNA hybrid branched nanomotifs (tiles), which readily assemble into one-dimensional nanofibres, extended two-dimensional arrays and a discrete three-dimensional object. The current strategy could enable the integration of the precise programmability of DNA with the rich functionality of RNA.

Ko, Seung Hyeon; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

2010-01-01

26

Synergistic self-assembly of RNA and DNA molecules  

NASA Astrophysics Data System (ADS)

DNA has recently been used as a programmable 'smart' building block for the assembly of a wide range of nanostructures. It remains difficult, however, to construct DNA assemblies that are also functional. Incorporating RNA is a promising strategy to circumvent this issue as RNA is structurally related to DNA but exhibits rich chemical, structural and functional diversities. However, only a few examples of rationally designed RNA structures have been reported. Herein, we describe a simple, general strategy for the de novo design of nanostructures in which the self-assembly of RNA strands is programmed by DNA strands. To demonstrate the versatility of this approach, we have designed and constructed three different RNA-DNA hybrid branched nanomotifs (tiles), which readily assemble into one-dimensional nanofibres, extended two-dimensional arrays and a discrete three-dimensional object. The current strategy could enable the integration of the precise programmability of DNA with the rich functionality of RNA.

Ko, Seung Hyeon; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

2010-12-01

27

DNA Virus MicroRNA and Methods for Inhibiting Same.  

National Technical Information Service (NTIS)

The invention relates to isolated nucleic acid molecules comprising the sequence of a human cytomegalovirus microRNA. In another embodiment, the invention relates to single stranded DNA virus microRNA molecules comprising the sequence of a human cytomegal...

S. Pfeffer T. Tuschl

2004-01-01

28

Modulation of biological processes by mineral fiber adsorption of macromolecules in vitro.  

PubMed

Three classes of macromolecules (i.e., DNA, RNA, and protein) were shown to be adsorbed to asbestiform minerals. The cytotoxicity exerted by the fibers on a normal human fibroblast cell line, which may be an indicator of the carcinogenic potential of mineral fibers, correlated positively with the degree of macromolecular adsorption of the fiber, namely: chrysotile greater than amosite greater than glass fiber. Asbestiform fibers also induce an alteration in in vitro DNA hydrolysis by bovine pancreatic deoxyribonuclease. This phenomenon suggests that adsorption by asbestiform minerals may modulate biological processes by inducing a conformational change in biological macromolecules as a result of coulombic interaction between the surface charge of the fiber and the hydrophilic groups on the macromolecule. PMID:1700100

Chang, M J; Joseph, L B; Stephens, R E; Hart, R W

29

Widespread RNA and DNA Sequence Differences in the Human Transcriptome  

PubMed Central

The transmission of information from DNA to RNA is a critical process. We compared RNA sequences from human B cells of 27 individuals to the corresponding DNA sequences from the same individuals and uncovered more than 10,000 exonic sites where the RNA sequences do not match that of the DNA. All 12 possible categories of discordances were observed. These differences were nonrandom as many sites were found in multiple individuals and in different cell types, including primary skin cells and brain tissues. Using mass spectrometry, we detected peptides that are translated from the discordant RNA sequences and thus do not correspond exactly to the DNA sequences. These widespread RNA-DNA differences in the human transcriptome provide a yet unexplored aspect of genome variation.

Li, Mingyao; Wang, Isabel X.; Li, Yun; Bruzel, Alan; Richards, Allison L.; Toung, Jonathan M.; Cheung, Vivian G.

2011-01-01

30

Transfer RNA genes of Euglena gracilis chloroplast DNA  

Microsoft Academic Search

Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino

Richard B. Hallick; Margaret J. Hollingsworth; Jac A. Nickoloff

1984-01-01

31

Conserved tRNA gene cluster in starfish mitochondrial DNA  

Microsoft Academic Search

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

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

1989-01-01

32

Mobile Macromolecules in Plant Development  

Microsoft Academic Search

Plant cells transmit developmental signals and distribute nutrients via dynamic intercellular channels termed plasmodesmata\\u000a (PD). Multidisciplinary inquiries have provided evidence that plasmodesmatal regulation is critical to fundamental plant functions,\\u000a such as development, host–pathogen interactions, and systemic RNA silencing. This review focuses on macromolecules that transport\\u000a cell-to-cell through PD and describes their implications on plant development.

Insoon Kim; Hyun-Sook Pai

2009-01-01

33

DNA Virus MicoRNA and Methods for Inhibiting Same.  

National Technical Information Service (NTIS)

The invention relates to isolated nucleic acid molecules comprising the sequence of any one of the DNA virus microRNAs shown in Table A1. In another embodiment, the invention relates to single stranded DNA virus microRNA molecules and anti-DNA virus micro...

S. Pfelfer T. H. Tuschl

2004-01-01

34

Transcription of BK virus DNA by Escherichia coli RNA polymerase: size and sequence analysis of RNA.  

PubMed Central

Transcription of human papovarirus BK superhelical DNA by Escherichia coli RNA polymerase yielded symmetric RNA with an average chain length of 1,3000 nucleotides. All regions of human papovavirus BK DNA were equally transcribed. At least four initiation sites were available to the procaryotic enzyme.

Meneguzzi, G; Milanesi, G

1978-01-01

35

The chemical stability of abasic RNA compared to abasic DNA.  

PubMed

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2'-O-protecting group together with the analogous DNA and the 2'-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2'-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37 degrees C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via beta,delta-elimination and 2',3'-cyclophosphate formation. We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role. PMID:17151071

Küpfer, Pascal A; Leumann, Christian J

2006-12-06

36

Supporting the design of efficient dendritic DNA and siRNA nano-carriers with molecular modeling.  

PubMed

The design of macromolecules able to generate a stable binding with nucleic acids is of great interest for their possible application in gene delivery. During the last years particular attention has been addressed to the use of dendritic scaffolds as a base to construct efficient DNA and siRNA nano-carriers. Dendrimers and dendrons are hyperbranched polymers characterized by a well-defined structure and by the possibility to functionalize their surface in many different ways. In particular, their multivalent character allows the creation of multiple binding sites between the positively charged groups that decorate the surface of cationic dendrons and dendrimers and the negatively charged phosphate groups present on the strands of DNA and siRNA. The engineering of "ideal dendritic candidates" to deliver and release genetic materials into cells is, however, not trivial due to the huge distance that exists between the design phase and the real application of such molecules. A different architecture of the dendritic scaffold (flexible or rigid) can strongly modify the binding efficiency, but, at the same time, is influenced by the interactions with the external solution. In this context, molecular simulation can represent a "virtual bridge" between the design and the comprehension of the real behavior of such macromolecules. PMID:21745180

Pavan, Giovanni M; Danani, Andrea

2011-12-01

37

RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.  

PubMed Central

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.

Brabant, F; Acheson, N H

1995-01-01

38

Relative Rates of Retroviral Reverse Transcriptase Template Switching during RNA and DNA-Dependent DNA Synthesis  

Microsoft Academic Search

Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp

ROBERT R. BOWMAN; WEI-SHAU HU; VINAY K. PATHAK; Mary Babb

1998-01-01

39

RNA polymerase induces DNA bending at yeast mitochondrial promoters.  

PubMed Central

Yeast mitochondrial RNA polymerase can bind specifically to promoter-containing DNA fragments in vitro as detected by DNAse I or methidiumpropyl-EDTA. Fe(II) protection assays and gel retardation experiments. Retardation of RNA polymerase-DNA complexes was most pronounced when the promoter was located in the middle of a DNA fragment and was diminished when RNA polymerase was bound near one of the ends. This indicates that upon RNA polymerase-binding the DNA undergoes a conformational change which is most likely a bend. The degree of introduced bending correlated with the efficiency of transcription and promoter-binding in a series of promoter mutants, suggesting that bending is a functional event during promoter utilisation. Images

Schinkel, A H; Groot Koerkamp, M J; Teunissen, A W; Tabak, H F

1988-01-01

40

Dynamic Methods for Investigating the Conformational Changes of Biological Macromolecules  

NASA Astrophysics Data System (ADS)

Fast conformational changes of biological macromolecules such as RNA folding and DNA-protein interactions play a crucial role in their biological functions. Conformational changes are supposed to take place in the sub milliseconds to few seconds time range. The development of appropriate dynamic methods possessing both high space (one nucleotide) and time resolution is of important interest. Here, we present two different approaches we developed for studying nucleic acid conformational changes such as salt-induced tRNA folding and interaction of the transcription factor NF-?B with its recognition DNA sequence. Importantly, only a single laser pulse is sufficient for the accurate measuring the whole decay curve. This peculiarity can be used in dynamical experiments.

Vidolova-Angelova, E.; Peshev, Z.; Shaquiri, Z.; Angelov, D.

2010-01-01

41

Localization of DNA and RNA in Eosinophil Secretory Granules  

Microsoft Academic Search

Background: Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. Methods: We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine

Ali R. Behzad; David C. Walker; Thomas Abraham; John McDonough; Salahadin Mahmudi-Azer; Fanny Chu; Furquan Shaheen; James C. Hogg; Peter D. Paré

2010-01-01

42

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

43

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

44

A hairpin near the 5' end stabilises the DNA gyrase mRNA in Mycobacterium smegmatis  

Microsoft Academic Search

RNA is amongst the most labile macromolecules present in the cells. The steady-state levels of mRNA are regulated both at the stages of synthesis and degradation. Recent work in Escherichia coli suggests that controlling the rate of degradation is as important as the process of synthesis. The stabil- ity of mRNA is probably more important in slow- growing organisms like

Shyam Unniraman; Monalisa Chatterji; Valakunja Nagaraja

2002-01-01

45

Differential biophysical behavior of human telomeric RNA and DNA quadruplex.  

PubMed

Quadruplex forming potential has also been observed at the RNA level and has been associated with molecular function and recognition; however, biophysical characterization of these structures to understand their recognition properties still remains a subject of investigation. We employed CD and UV spectroscopy to understand the conformation and stability of a 24-mer human telomeric RNA quadruplex and compared it with that of the corresponding DNA analogue in 100 mM KCl containing solution. We observed that the RNA quadruplex adopts a parallel conformation and displays remarkably high thermal stabilities as compared to its DNA counterpart. Using the osmotic stress method, we determined the net release of 6.0+/-0.5 and 4.0+/-0.4 water molecules from the RNA and DNA quadruplex structure, respectively. Furthermore, UV titration experiments showed that both human telomeric RNA and DNA quadruplexes possess similar recognition abilities for TMPyP4 molecules. Our study provides a lead in extending the knowledge generated for the DNA quadruplex to the RNA level. PMID:19572668

Arora, Amit; Maiti, Souvik

2009-07-30

46

Phosphate-Methylated DNA Aimed at HIV1 RNA Loops and Integrated DNA Inhibits Viral Infectivity  

Microsoft Academic Search

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Both antisense and sense phosphate-methylated DNA 20-nucleotide oligomers, targeted at the

Henk M. Buck; Leo H. Koole; Marcel H. P. van Genderen; Lia Smit; Jan L. M. C. Geelen; Suzanne Jurriaans; Jaap Goudsmit

1990-01-01

47

Search for Scrapie-specific RNA and Attempts to Detect an Infectious DNA or RNA  

Microsoft Academic Search

SUMMARY Preparations of in vivo labelled RNA from normal and scrapie infected mouse brains were fractionated by polyacrylamide gel electrophoresis. No appreciable differences were detected between the two preparations. The biological activity of preparations of RNA and DNA from scrapie brain was also examined. In each case the nucleic acid was obtained by one method involving the use of phenol

G. D. Hunter; S. C. Collis; G. C. Millson; R. H. Kimberlin

1976-01-01

48

RNA recognition by the DNA end-binding Ku heterodimer.  

PubMed

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer. PMID:23610127

Dalby, Andrew B; Goodrich, Karen J; Pfingsten, Jennifer S; Cech, Thomas R

2013-04-22

49

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

50

a Hypothetical Pathway from the RNA to the DNA World  

NASA Astrophysics Data System (ADS)

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

Line, Martin A.

2005-08-01

51

RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions.  

PubMed Central

Rates of formation of RNA:DNA hybrids have been measured as a function of temperature and compared to DNA:RNA duplex denaturation temperatures in 4 M sodium perchlorate, 4 M NaClO4-6 M urea, and 3 M rubidium trichloracetate solvents. The usual bell shaped curves of reaction rate versus temperature were observed. The optimal temperatures for the RNA:DNA association reaction are 5 degrees to 12 degrees greater than the Tm's for DNA:DNA denaturation in these solvents, just as in formamide. R-loops of phi80d3ilv DNA with E. coli rRNA can be formed at high efficiency in these solvents. Images

Chien, Y H; Davidson, N

1978-01-01

52

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II  

Microsoft Academic Search

Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-]purin-10(3H)one (M1dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient

Susan D. Cline; James N. Riggins; Silvia Tornaletti; Lawrence J. Marnett; Philip C. Hanawalt

2004-01-01

53

Ribonucleic guanidine demonstrates an unexpected marked preference for complementary DNA rather than RNA  

Microsoft Academic Search

Replacement of the phosphodiester linkages of DNA and RNA by guanidinium linkages provides DNG and RNG. We report here the order of stability of mixed duplexes (RNG-U5·DNA-A5?RNA-U5·RNA-A5>RNG-U5·RNA-A5>RNA-U5·DNA-A5>DNA-T5·DNA-A5). The considerable stability of RNG·DNA compared to RNG·RNA is shown to be due to the rigid backbone of RNG existing only in B-form and therefore lowering its affinity for A-RNA. RNG oligomers are

Joseph W. Toporowski; Thomas C. Bruice

2006-01-01

54

Induction of fos RNA by DNA-damaging agents  

SciTech Connect

We have found that a wide variety of DNA-damaging agents and heat shock increase fos RNA to different extents in Chinese hamster ovary cells. These include the monofunctional alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, the cross-linking agents cis-Pt(II) diamminedichloride and mechlorethamine HCl, the DNA base-damaging agents 4-nitroquinoline-N-oxide and N-acetoxy-2-acetylaminofluorene, ultraviolet and near-ultraviolet radiation, hydrogen peroxide, and Adriamycin. Prolonged increases in fos RNA were observed, with persistence to 16 h following treatment. Variation in the amount of fos RNA induction by these agents and the diverse types and frequencies of cellular damage produced by them suggested that there might be several different mechanisms responsible for increased fos RNA.

Hollander, M.C.; Fornace, A.J. Jr.

1989-04-01

55

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: Complex theoretical and experimental studies.  

PubMed

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. PMID:23968861

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Spérová, Miroslava; Schneider, Bohdan; Páv, Ond?ej; Sebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-07-31

56

Simultaneous DNA and RNA isolation from brain punches for epigenetics  

PubMed Central

Background Epigenetic modifications such as DNA methylation play an important role for gene expression and are regulated by developmental and environmental signals. DNA methylation typically occurs in a highly tissue- and cell-specific manner. This raises a severe challenge when studying discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Because gene expression and methylation are often tightly linked it appears of interest to compare both parameters in the same sample. Findings We present a refined method for the simultaneous extraction of DNA for bisulfite sequencing and RNA for expression analysis from small mouse brain tissue punches. This method can also be easily adapted for other small tissues or cell populations. Conclusions The method described herein results in DNA and RNA of a quantity and quality permitting highly reliable bisulfite analysis and quantitative RT-PCR measurements, respectively.

2011-01-01

57

RNA Splicing: A New Player in the DNA Damage Response  

PubMed Central

It is widely accepted that tumorigenesis is a multistep process characterized by the sequential accumulation of genetic alterations. However, the molecular basis of genomic instability in cancer is still partially understood. The observation that hereditary cancers are often characterized by mutations in DNA repair and checkpoint genes suggests that accumulation of DNA damage is a major contributor to the oncogenic transformation. It is therefore of great interest to identify all the cellular pathways that contribute to the response to DNA damage. Recently, RNA processing has emerged as a novel pathway that may contribute to the maintenance of genome stability. In this review, we illustrate several different mechanisms through which pre-mRNA splicing and genomic stability can influence each other. We specifically focus on the role of splicing factors in the DNA damage response and describe how, in turn, activation of the DDR can influence the activity of splicing factors.

Lenzken, Silvia C.; Barabino, Silvia M. L.

2013-01-01

58

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.

Pooggin, Mikhail M.

2013-01-01

59

RNA-DNA Interactions and DNA Methylation in PostTranscriptional Gene Silencing  

Microsoft Academic Search

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

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

1999-01-01

60

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

61

Comparison of DNA and RNA extraction methods for mummified tissues  

Microsoft Academic Search

Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were

Nami Konomi; Eve Lebwohl; David Zhang

2002-01-01

62

Prospects of chimeric RNA-DNA oligonucleotides in gene therapy  

Microsoft Academic Search

A strategy called targeted gene repair was developed to facilitate the process of gene therapy using a chimeric RNA-DNA oligonucleotide. Experiments demonstrated the feasibility of using the chimeric oligonucleotide to introduce point conversion in genes in vitro and in vivo. However, barriers exist in the low and\\/or inconstant frequency of gene repair. To overcome this difficulty, three main aspects should

Xue-Song Wu; De-Pei Liu; Chih-Chuan Liang

2001-01-01

63

Antibody to the RNA-Dependent DNA Polymerase of Mammalian C-Type RNA Tumor Viruses  

Microsoft Academic Search

Sera from rats bearing transplantable tumors induced by murine C-type tumor viruses contain an inhibitor of the activity of the viral RNA-dependent DNA polymerase. The inhibitor is shown to be an immunoglobulin (IgG) directed against the enzyme. Antiserum made in rabbits against partially purified murine leukemia virus polymerase also inhibits the polymerases of other mammalian C-type RNA-containing tumor viruses. Thus,

Stuart A. Aaronson; Wade P. Parks; Edward M. Scolnick; George J. Todaro

1971-01-01

64

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

65

A superfamily of DNA transposons targeting multicopy small RNA genes.  

PubMed

Target-specific integration of transposable elements for multicopy genes, such as ribosomal RNA and small nuclear RNA (snRNA) genes, is of great interest because of the relatively harmless nature, stable inheritance and possible application for targeted gene delivery of target-specific transposable elements. To date, such strict target specificity has been observed only among non-LTR retrotransposons. We here report a new superfamily of sequence-specific DNA transposons, designated Dada. Dada encodes a DDE-type transposase that shows a distant similarity to transposases encoded by eukaryotic MuDR, hAT, P and Kolobok transposons, as well as the prokaryotic IS256 insertion element. Dada generates 6-7 bp target site duplications upon insertion. One family of Dada DNA transposons targets a specific site inside the U6 snRNA genes and are found in various fish species, water flea, oyster and polycheate worm. Other target sequences of the Dada transposons are U1 snRNA genes and different tRNA genes. The targets are well conserved in multicopy genes, indicating that copy number and sequence conservation are the primary constraints on the target choice of Dada transposons. Dada also opens a new frontier for target-specific gene delivery application. PMID:23874566

Kojima, Kenji K; Jurka, Jerzy

2013-07-09

66

Analysis of macromolecules, ligands and macromolecule-ligand complexes  

SciTech Connect

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

67

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

PubMed

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

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

2006-01-01

68

Monomers in coal macromolecules  

Microsoft Academic Search

The objective of this study is to elucidate the nature of the thermally stable monomers released from coal macromolecules upon heating. Although these species represent only a portion of the coal structure which varies with rank, these molecules pay important roles in a variety of coal reactions. These reactions include: pyrolysis, hydropyrolysis, and the early stages of liquefaction, gasification and

R. E. Winans; R. L. McBeth; J. E. Hunt; P. E. Melnikov

1991-01-01

69

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

70

Single-molecule electrical random resequencing of DNA and RNA.  

PubMed

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. PMID:22787559

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

2012-07-10

71

[Spin polymerization of DNA/RNA nucleotides].  

PubMed

The Car-Parrinello molecular dynamics (CPMD) has been used to study the ion-radical (IR) polymerization (triplet (T) and singlet (S/TO) states) of adenine mononucleotides upon interaction with Mg2+(H2O)2-ATP(4-). It has been found that the IR polymerization occurs only upon Mg2+(H2O)2-ATP(4-) excitation into a T state (the Franck-Condon or femtosecond laser excitation); it naturally occurs in the dark with DNA polymerase or another Mg-holoenzyme upon interaction of Mg with two Asp residues. The IR path affects only the HO-C3' group of ribose, leaving the HO-C2' group inactive. The IR polymerization starts with the homolytic removal of the hydrogen atom from the HO-C3' group and its transfer onto the hydroxyl radical *OH, a product of the ATP cleavage, which yields a water molecule. A further progress of the reaction involves interaction between two ion-radicals *AMP. The reaction is sensitive to the recombination of *OH and *AMP. It is mostly suppressed by the appearance of identically directed electron spins on both radicals (the radical pair in the T-state) in the vicinity of the HO-C3' group and not suppressed in the vicinity of the HO-C2' group (the spins in the radical pair are oppositely directed, the radical pair in the To state), making the latter inert on the IR polymerization, but allowing it to be active in the ionic (hydrolytic) polymerization. PMID:21542349

Tulub, A A

72

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.  

PubMed Central

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.

Fujiwara, T; Giesman-Cookmeyer, D; Ding, B; Lommel, SA; Lucas, WJ

1993-01-01

73

Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.  

PubMed Central

A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 9 FIGURE 12 FIGURE 13

Hummer, G; Garcia, A E; Soumpasis, D M

1995-01-01

74

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP  

Microsoft Academic Search

Background: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP)' with micromolar affinity exhibit distinct binding site sequences and secondary structures. We

Chin H. Lin; Dinshaw J. Patei

1997-01-01

75

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.  

PubMed Central

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

Liquier, J; Taillandier, E; Klinck, R; Guittet, E; Gouyette, C; Huynh-Dinh, T

1995-01-01

76

A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.  

PubMed Central

In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed.

Pelissier, T; Wassenegger, M

2000-01-01

77

RNA mapping on Drosophila mitochondrial DNA: precursors and template strands.  

PubMed Central

Drosophila melanogaster mitochondrial DNA (mtDNA) is closely related to the mammalian and amphibian mtDNA except for gene organization. In Drosophila, genes are distributed in clusters alternatively coded on each strand. Besides the eleven major foreseeable transcripts previously described (MERTEN and PARDUE, 1981, J. Mol. Biol., 153, 1-21), we have characterized two poly A+ transcripts, one major and one minor which could correspond respectively to the ND3 and ND6 reading frames, and 27 poly A+ minor transcripts (0.2 to greater than 3.2 kb) which are distributed along the mtDNA except in the rRNAs, ND 1 and A+ T rich regions. The mapping and length of 25 of these transcripts strongly suggest a precursor role. They would be processed at the level of tRNA or tRNA-like sequences. Most of them are transcribed from the template strand of each gene cluster and their distribution is in agreement with the hypothesis of several transcription origins and terminations located near the extremities of each gene cluster. Quantitatively our results show a large variation in each presumptive mature transcript compared to the other, even in a given gene cluster, suggesting a specific degradation of some of the mature transcripts. Images

Berthier, F; Renaud, M; Alziari, S; Durand, R

1986-01-01

78

Coupling ? Factor Conformation to RNA Polymerase Reorganisation for DNA Melting  

PubMed Central

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (?54-RNAP, E?54) and a slowly hydrolysed ATP analogue (ATP?S), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of ?54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of E?54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity ?54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic ?/?’ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for E?54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the ?’ jaw domain, thereby allowing acquisition of the open complex status.

Burrows, Patricia C.; Joly, Nicolas; Cannon, Wendy V.; Camara, Beatriz P.; Rappas, Mathieu; Zhang, Xiaodong; Dawes, Kathleen; Nixon, B. Tracy; Wigneshweraraj, Siva R.; Buck, Martin

2009-01-01

79

Intergenic transcripts originating from a subclass of ribosomal DNA repeats silence ribosomal RNA genes in trans.  

PubMed

Epigenetic silencing of a fraction of ribosomal DNA (rDNA) requires association of the nucleolar chromatin-remodelling complex NoRC to 150-250 nucleotide RNAs (pRNA) that originate from an RNA polymerase I promoter located in the intergenic spacer separating rDNA repeats. Here, we show that NoRC-associated pRNA is transcribed from a sub-fraction of hypomethylated rRNA genes during mid S phase, acting in trans to inherit DNA methylation and transcriptional repression of late-replicating silent rDNA copies. The results reveal variability between individual rDNA clusters with distinct functional consequences. PMID:20010804

Santoro, Raffaella; Schmitz, Kerstin-Maike; Sandoval, Juan; Grummt, Ingrid

2009-12-04

80

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.  

PubMed

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage. PMID:23775790

Calkins, Anne S; Iglehart, J Dirk; Lazaro, Jean-Bernard

2013-06-17

81

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1  

PubMed Central

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

Calkins, Anne S.; Iglehart, J. Dirk; Lazaro, Jean-Bernard

2013-01-01

82

DNA and RNA-based vaccines: principles, progress and prospects  

PubMed Central

DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), co-stimulatory molecules and cytokine- and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them ‘self-replicating’. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 ?g of nucleic acid injected once intramuscularly. Cells transfected with ‘self-replicating’ vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of ‘self-replicating’ genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

Leitner, Wolfgang W.; Ying, Han; Restifo, Nicholas P.

2007-01-01

83

Chimeric RNA–DNA molecular beacon assay for ribonuclease H activity  

Microsoft Academic Search

Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming. Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids. An RNA–DNA chimeric beacon assay for RNase H enzymatic activity was developed. The substrate is a single-stranded RNA–DNA chimeric oligonucleotide labeled

J. Rizzo; L. K. Gifford; X. Zhang; A. M. Gewirtz; P. Lu

2002-01-01

84

Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA  

Microsoft Academic Search

Telomerase is a specialized DNA polymerase that extends the 3? ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical

Meghan Mitchell; Andrew Gillis; Mizuko Futahashi; Haruhiko Fujiwara; Emmanuel Skordalakes

2010-01-01

85

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.  

PubMed Central

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.

de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

1988-01-01

86

Magnetic nanoparticle formulations for DNA and siRNA delivery  

NASA Astrophysics Data System (ADS)

Newly synthesized magnetic nanomaterials possess high DNA binding capacity either itself or in the presence of a positively charged lipid-based Metafectene™ reagent or branched polyethylene imine 25 kDa. Polyethylene imine (PEI)-modified nanomaterials are able to deliver nucleic acids in cell culture in duplexes. Magnetofection with triplexes of nanomaterials results in higher transduction efficiencies compared to optimal PEI or Metafectene formulations. 90% transient down-regulation of the target protein in HeLa-green fluorescence protein cells was achieved at short interfering RNA concentrations as low as 8 nM with a formulation of PEI-modified nanoparticles.

Mykhaylyk, Olga; Vlaskou, Dialekti; Tresilwised, Nittaya; Pithayanukul, Pimolpan; Möller, Winfrid; Plank, Christian

2007-04-01

87

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

2013-07-10

88

RNA aptamers selected against DNA polymerase b inhibit the polymerase activities of DNA polymerases b and k  

Microsoft Academic Search

DNA polymerase b (polb), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polb from a variable pool of 8 3 1012 individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened

Leonid V. Gening; Svetlana A. Klincheva; Anastasia Reshetnjak; Arthur P. Grollman; Holly Miller

2006-01-01

89

DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation.  

PubMed

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA-RNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNA-RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNA-RNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNA-RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA-RNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA-RNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNA-RNA hybrid formation plays a role in chromatic ncRNA function. PMID:22280061

Nakama, Mina; Kawakami, Kei; Kajitani, Takuya; Urano, Takeshi; Murakami, Yota

2012-01-27

90

Deploying RNA and DNA with Functionalized Carbon Nanotubes.  

PubMed

Carbon nanotubes internalize into cells and are potential molecular platforms for siRNA and DNA delivery. A comprehensive understanding of the identity and stability of ammoniumfunctionalized carbon nanotube (f-CNT)-based nucleic acid constructs is critical to deploying them in vivo as gene delivery vehicles. This work explored the capability of f-CNT to bind single- and double-strand oligonucleotides by determining the thermodynamics and kinetics of assembly and the stoichiometric composition in aqueous solution. Surprisingly, the binding affinity of f-CNT and short oligonucleotide sequences was in the nanomolar range, kinetics of complexation were extremely rapid, and from one to five sequences were loaded per nanotube platform. Mechanistic evidence for an assembly process that involved electrostatic, hydrogen-bonding and ?-stacking bonding interactions was obtained by varying nanotube functionalities, oligonucleotides, and reaction conditions. (31)P-NMR and spectrophotometric fluorescence emission data described the conditions required to assemble and stably bind a DNA or RNA cargo for delivery in vivo and the amount of oligonucleotide that could be transported. The soluble oligonucleic acid-f-CNT supramolecular assemblies were suitable for use in vivo. Importantly, key evidence in support of an elegant mechanism by which the bound nucleic acid material can be 'off-loaded' from the f-CNT was discovered. PMID:23626864

Alidori, Simone; Asqiriba, Karim; Londero, Pablo; Bergkvist, Magnus; Leona, Marco; Scheinberg, David A; McDevitt, Michael R

2013-03-21

91

DNA methylation mediated by a microRNA pathway.  

PubMed

In plants, the known microRNAs (miRNAs) are produced as approximately 21 nucleotide (nt) duplexes from their precursors by Dicer-like 1 (DCL1). They are incorporated into Argonaute 1 (AGO1) protein to regulate target gene expression primarily through mRNA cleavage. We report here the discovery of a class of miRNAs in the model monocot rice (Oryza sativa). These are 24 nt in length and require another member of the Dicer family, DCL3, for their biogenesis. The 24 nt long miRNAs (lmiRNAs) are loaded into AGO4 clade proteins according to hierarchical rules, depending on the upstream biogenesis machinery and the 5'-terminal nucleotide. We demonstrated that lmiRNAs direct DNA methylation at loci from which they are produced as well as in trans at their target genes and play roles in gene regulation. Considered together, our findings define a miRNA pathway that mediates DNA methylation. PMID:20381393

Wu, Liang; Zhou, Huanyu; Zhang, Qingqing; Zhang, Jianguang; Ni, Fangrui; Liu, Chang; Qi, Yijun

2010-04-08

92

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

SciTech Connect

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

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

2010-01-01

93

Coarse-grained simulations of RNA and DNA duplexes.  

PubMed

Although RNAs play many cellular functions, little is known about the dynamics and thermodynamics of these molecules. In principle, all-atom molecular dynamics simulations can investigate these issues, but with current computer facilities, these simulations have been limited to small RNAs and to short times. HiRe-RNA, a recently proposed high-resolution coarse-grained RNA that captures many geometric details such as base pairing and stacking, is able to fold RNA molecules to near-native structures in a short computational time. So far, it had been applied to simple hairpins, and here we present its application to duplexes of a couple dozen nucleotides and show how with replica exchange molecular dynamics (REMD) we can easily predict the correct double helix from a completely random configuration and study the dissociation curve. To show the versatility of our model, we present an application to a double stranded DNA molecule as well. A reconstruction algorithm allows us to obtain full atom structures from the coarse-grained model. Through atomistic molecular dynamics (MD), we can compare the dynamics starting from a representative structure of a low temperature replica or from the experimental structure, and show how the two are statistically identical, highlighting the validity of a coarse-grained approach for structured RNAs and DNAs. PMID:23730911

Cragnolini, Tristan; Derreumaux, Philippe; Pasquali, Samuela

2013-06-26

94

Characterization of a novel RNA polymerase II arrest site which lacks a weak 3' RNA-DNA hybrid.  

PubMed

Transcript elongation by RNA polymerase II is blocked at DNA sequences called arrest sites. An exceptionally weak RNA-DNA hybrid is often thought to be necessary at the point of arrest. We have identified an arrest site from the tyrosine hydroxylase (TH) gene which does not fit this pattern. Transcription of many sequence variants of this site shows that the RNA-DNA hybrid over the three bases immediately preceding the major arrest point may be strong (i.e. C:G) without interfering with arrest. However, arrest at the TH site requires the presence of a pyrimidine at the 3' end and arrest increases when the 3'-most segment is pyrimidine rich. We also demonstrated that arrest at the TH site is completely dependent on the presence of a purine-rich element immediately upstream of the RNA-DNA hybrid. Thus, the RNA polymerase II arrest element from the TH gene has several unanticipated characteristics: arrest is independent of a weak RNA-DNA hybrid at the 3' end of the transcript, but it requires both a pyrimidine at the 3' end and a polypurine element upstream of the RNA-DNA hybrid. PMID:15047857

Hawryluk, Peter J; Ujvári, Andrea; Luse, Donal S

2004-03-26

95

Unusual sequences, homologous to 5S RNA, in ribosomal DNA repeats of the nematode Meloidogyne arenaria  

Microsoft Academic Search

There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematodeMeloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and

Haleh Vahidi; John Curran; Donald W. Nelson; John M. Webster; Michael A. McClure; Barry M. Honda

1988-01-01

96

A comparison of DNA and RNA-based clone libraries from the same marine bacterioplankton community  

Microsoft Academic Search

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by ?-Proteobacteria in terms of repetitive DNA clones (52%), but ?-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized

Markus M. Moeseneder; Jesus M. Arrieta; Gerhard J. Herndl

2005-01-01

97

Isolation of a cDNA clone for human threonyl-tRNA synthetase  

SciTech Connect

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA /lambda/gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Kontis, K.J.; Arfin, S.M.

1989-05-01

98

RNA polymerase I catalysed transcription of insert viral cDNA.  

PubMed Central

RNA polymerase I has been used for transcription of influenza hemagglutinin (HA) cDNA precisely linked in the anti-sense configuration to both mouse rDNA promoter and terminator segments. In transcription reactions based on Ehrlich ascites cell nuclear extracts, specific uniform RNA products are synthesized in high rates that are comparable to original rDNA template transcriptions. Primer extension reactions show the 5' ends of these RNA transcripts to be located exactly at position +1, corresponding to the 5' end of negative strand HA viral RNA. RNA 3' ends in a first series of constructs were found extended beyond the accepted location of pre-rRNA 3' ends, in using both hybrid cDNA and original rDNA templates. But upon deletion of six basepairs from the rDNA termination region RNA polymerase I transcription has been adapted to yield correctly terminated influenza viral RNA in vitro. This result has been confirmed in an in vivo experiment via synthesis of an anti-sense viral RNA molecule containing the chloramphenicol acetyltransferase (CAT) gene, which in turn is recognized at its terminal sequence by viral RNA dependent RNA polymerase for plus strand mRNA synthesis and expression of CAT activity. Images

Zobel, A; Neumann, G; Hobom, G

1993-01-01

99

Rapid and Reliable Method of Extracting DNA and RNA from Sweetpotato, Ipomoea batatas (L). Lam  

Microsoft Academic Search

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg\\/g leaf tissue). The extracted DNA was completely digested by restriction

Sun-Hyung Kim; Tatsuro Hamada

2005-01-01

100

Ancient RNA? RT-PCR of 50-year-old RNA identifies peach latent mosaic viroid.  

PubMed

The preservation of macromolecules is at best haphazard. Modern techniques have improved the detection of ancient DNA and proteins, but there is little information on the preservation of RNA. Fifty-year-old dried leaf material showing symptoms of peach calico disease was used successfully in RT-PCRs to amplify peach latent mosaic viroid (PLMVd) RNA and the mRNA for the large subunit of ribulose 1,5-bisphosphate carboxylase (rubisco). These results indicate that naked RNA may be preserved, under suitable conditions, for at least 50 years. The results are discussed in the context of ancient DNA and proteins and the process of fossilization. PMID:23138153

Guy, Paul L

2012-11-09

101

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

PubMed

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

2013-10-04

102

Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA.  

PubMed

Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans. PMID:1276156

Garrett, C T; McNulty, M E

1976-06-01

103

First-principles GW calculations for DNA and RNA nucleobases  

NASA Astrophysics Data System (ADS)

On the basis of first-principles GW calculations, we study the quasiparticle properties of the guanine, adenine, cytosine, thymine, and uracil DNA and RNA nucleobases. Beyond standard G0W0 calculations, starting from Kohn-Sham eigenstates obtained with (semi)local functionals, a simple self-consistency on the eigenvalues allows us to obtain vertical ionization energies and electron affinities within an average 0.11 and 0.18 eV error, respectively, as compared to state-of-the-art coupled-cluster and multiconfigurational perturbative quantum chemistry approaches. Further, GW calculations predict the correct ?-character of the highest occupied state, due to several level crossings between density functional and GW calculations. Our study is based on a recent Gaussian-basis implementation of GW calculations with explicit treatment of dynamical screening through contour deformation techniques.

Faber, Carina; Attaccalite, Claudio; Olevano, V.; Runge, E.; Blase, X.

2011-03-01

104

Effects of DNA lesions on the transcription reaction of mitochondrial RNA polymerase: implications for bypass RNA synthesis on oxidative DNA lesions.  

PubMed

Oxidative DNA lesions inhibit the transcription of RNA polymerase II, but in the presence of transcription elongation factors, the transcription can bypass the lesions. Single-subunit mitochondrial RNA polymerase (mtRNAP) catalyses the synthesis of essential transcripts in mitochondria where reactive oxidative species (ROS) are generated as by-products. The occurrence of RNA synthesis by mtRNAP at oxidative DNA lesions remains unknown. Purified mtRNAP and a complex of RNA primer/DNA template containing a single DNA lesion, such as ROS-induced 8-oxoguanine (8-oxoG), two isomeric thymine glycols (5R-Tg or 5S-Tg), the UV-induced cis-syn cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone photoproduct (6-4pp), or a spontaneous common DNA lesion, a base-loss-induced apurinic/apyrimidinic (AP) site, were used for in vitro RNA synthesis assays. In this report, we show that mtRNAP bypassed the oxidative DNA lesions of non-bulky 8-oxoG and 5R-Tg and 5S-Tg with pausing sites but did not bypass the UV-induced DNA lesions and the AP site. The bacteriophage T7 phage RNA polymerase, which is homologous to mtRNAP, bypassed 8-oxoG but stalled at 5R-Tg and 5S-Tg. As expected, although translesion RNA synthesis in 8-oxoG on the DNA templates generated incorrect transcripts with a G:C to T:A transversion, the synthesis in Tg could lead to the correct transcripts with no transcriptional mutagenesis. Collectively, these data suggest that mtRNAP may tolerate the mitochondrial genome containing oxidative DNA lesions induced by ROS from the side effects of an ATP generation reaction. PMID:23053822

Nakanishi, Nozomi; Fukuoh, Atsushi; Kang, Dongchon; Iwai, Shigenori; Kuraoka, Isao

2012-10-10

105

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA  

PubMed Central

RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5?-PO4 single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65°C, AppRNA was predominantly ligated to a circular product. In contrast, at 35°C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases.

Torchia, Christopher; Takagi, Yuko; Ho, C. Kiong

2008-01-01

106

Chromatin-bound DNA-dependent RNA polymerase in developing pea cotyledons  

Microsoft Academic Search

The pattern of cotyledon development in three varieties of Pisum sativum has been defined in terms of cell number, DNA and RNA content and chromatin, bound RNA polymerase activity. Variation was observed in the relative periods of growth by cell division and cell expansion between the three varieties. The mean DNA content per cotyledon cell during growth by cell expansion

C. A. Cullis

1976-01-01

107

Aflatoxin B1: Binding to DNA in vitro and Alteration of RNA Metabolism in vivo  

Microsoft Academic Search

Aflatoxin B1 binds to both native and denatured DNA, as shown by spectroscopy and equilibrium dialysis. It also strongly inhibits incorporation of cytidine into rat liver nuclear RNA and lowers the RNA content of the nucleus. The extreme toxicity and carcinogenicity of aflatoxin B1 may be direct results of the affinity of this agent for DNA.

Michael B. Sporn; C. Wesley Dingman; Harriette Longacre Phelps; Gerald N. Wogan

1966-01-01

108

RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency  

PubMed Central

Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template-free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template-translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template-containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation.

Qi, Xiaodong; Xie, Mingyi; Brown, Andrew F; Bley, Christopher J; Podlevsky, Joshua D; Chen, Julian J-L

2012-01-01

109

RNA electrophoretic mobility shift assay using a fluorescent DNA sequencer  

Microsoft Academic Search

The RNA electrophoretic mobility shift assay is a simple and rapid method for visualizing the existence of specific RNA-protein interaction. We have developed a useful method for the detection of mRNA binding proteins using non-radioactive RNA. In this method, RNA was prepared by in vitro transcription using Texas Red-labeled nucleotides. The synthesized RNA from chicken aA-globin 3'-UTR region, which has been

Yukinori Eguchi; Tomoko Eguchi

2001-01-01

110

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

PubMed

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

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

2013-09-12

111

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

PubMed Central

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

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

2013-01-01

112

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

113

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.

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

2011-01-01

114

Thermodynamics and Kinetics of DNA, RNA, and Hybrid Oligonucleotide Double-Strand Formation.  

National Technical Information Service (NTIS)

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 D...

J. W. Nelson

1982-01-01

115

The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response  

PubMed Central

SUMMARY The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one fourth of miRNAs are significantly up-regulated after DNA damage, while loss of ATM abolishes their induction. KSRP (KH-type splicing regulatory protein) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.

Zhang, Xinna; Wan, Guohui; Berger, Franklin G.; He, Xiaoming; Lu, Xiongbin

2011-01-01

116

The ATM kinase induces microRNA biogenesis in the DNA damage response.  

PubMed

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis. PMID:21329876

Zhang, Xinna; Wan, Guohui; Berger, Franklin G; He, Xiaoming; Lu, Xiongbin

2011-02-18

117

tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.  

PubMed

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

2013-07-18

118

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.

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

2013-01-01

119

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

120

Concordance between HIV-2 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA.  

PubMed

In this study, assessing HIV-2 tropism among 43 paired plasma/peripheral blood mononuclear cell specimens, the concordance between proviral DNA and plasma RNA genotypic tropism prediction was 74%. All the discordances were attributable to the prediction of R5 in RNA and X4/dual-mixed in DNA. HIV-2 genotypic tropism test based on proviral DNA is a suitable tool for tropism determination in HIV-2-infected patients with low or undetectable viral load. PMID:23095312

Visseaux, Benoit; Charpentier, Charlotte; Taieb, Audrey; Damond, Florence; Bénard, Antoine; Larrouy, Lucile; Chęne, Genevičve; Brun-Vézinet, Françoise; Matheron, Sophie; Descamps, Diane

2013-01-14

121

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

NASA Astrophysics Data System (ADS)

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

Tripathi, Tripti; Chowdhury, Debashish

2008-01-01

122

EFFECT OF THE LAMBDA REPRESSOR ON THE BINDING OF RNA POLYMERASE TO DNA  

PubMed Central

Experiments were designed with the aim of understanding the mechanism whereby the lambda repressor inhibits messenger RNA synthesis. Complexes containing lambda DNA and RNA polymerase were isolated from repressed and derepressed lysogens and analyzed for the relative amounts of RNA polymerase per phage genome. The data indicate that the repressor inhibits the binding of RNA polymerase to promoter sites known to be under direct control of the repressor.

Hayward, William S.; Green, Melvin H.

1969-01-01

123

Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication  

Microsoft Academic Search

SUMMARY Mitochondrial DNA is replicated by a unique enzy- matic machinery, which is distinct from the replica- tion apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochon- drial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the

Javier Miralles Fuste ´; Sjoerd Wanrooij; Elisabeth Jemt; Caroline E. Granycome; Tricia J. Cluett; Yonghong Shi; Neli Atanassova; Ian J. Holt; Claes M. Gustafsson; Maria Falkenberg

2010-01-01

124

Detection of enteroviral RNA and specific DNA of herpesviruses by multiplex genome amplification  

Microsoft Academic Search

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification

I. Casas; A. Tenorio; J. M. Echevarria; P. E. Klapper; G. M. Cleator

1997-01-01

125

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

Microsoft Academic Search

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

Mariaclara Ańez-Lingerfelt; George E. Fox; Richard C. Willson

2009-01-01

126

Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases  

PubMed Central

Background The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. Results Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the ?' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi ?-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP ?' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases. In addition to the ?' subunit, ? subunit of DDRP also contains a DPBB domain, which is, however, distorted by large inserts and does not harbor a counterpart of the DbDGD motif. The DPBB domains of the two DDRP subunits together form the catalytic cleft, with the domain from the ?' subunit supplying the metal-coordinating DbDGD motif and the one from the ? subunit providing two lysine residues involved in catalysis. Given that the two DPBB domains of DDRP contribute completely different sets of active residues to the catalytic center, it is hypothesized that the ultimate ancestor of RNA polymerases functioned as a homodimer of a generic, RNA-binding DPBB domain. This ancestral protein probably did not have catalytic activity and served as a cofactor for a ribozyme RNA polymerase. Subsequent evolution of DDRP and RDRP involved accretion of distinct sets of additional domains. In the DDRPs, these included a RNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybrid motif (SBHM) domain. Further, lineage-specific accretion of SBHM domains and other, DDRP-specific domains is observed in bacterial DDRPs. In contrast, the orthologs of the ?' subunit in archaea and eukaryotes contains a four-stranded ? + ? domain that is shared with the ?-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11, translation factor eIF1 and type II topoisomerases. The additional domains of the RDRPs remain to be characterized. Conclusions Eukaryotic RNA-dependent RNA polymerases share the catalytic double-psi ?-barrel domain, containing a signature metal-coordinating motif, with the universally conserved ?' subunit of DNA-dependent RNA polymerases. Beyond this core catalytic domain, the two classes of RNA polymerases do not have common domains, suggesting early divergence from a common ancestor, with subsequent independent domain accretion. The ?-subunit of DDRP contains another, highly diverged DPBB domain. The presence of two distinct DPBB domains in two subunits of DDRP is compatible wit

Iyer, Lakshminarayan M; Koonin, Eugene V; Aravind, L

2003-01-01

127

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.  

PubMed

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli). PMID:23549043

Ordonez, Heather; Shuman, Stewart

2013-04-02

128

DNA Sequencing Research Group (DSRG): Evaluation of RNA Amplification Kits at Subnanogram Input Amounts of Total RNA for RNA-Seq  

PubMed Central

Multiple recent publications on RNA-Seq have demonstrated the power of next generation sequencing technologies in whole transcriptome analysis. The vendor specific protocols used for RNA library construction typically require at least 100ng of total RNA. However, under certain conditions such as single cells, stem cells, difficult to isolate cell types, or fractionated cancer cells, only a small amount of material is available. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several RNA amplification kits are available for amplification prior to library construction and next generation sequencing but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-Seq for picogram amounts of RNA. This study conducted by the DNA Sequencing Research Group (DSRG) focuses on the evaluation of amplification kits for RNA-Seq. Four commercial amplification kits were chosen: Ovation v2 (NuGEN Technologies), SMARTer (Clontech), Seqplex (Sigma Aldrich), and Super-AMP (Miltenyi Biotech). Starting material was 5ng, 500pg and 50pg of human total reference RNA (Clontech) spiked with Ambion ERCC control mix (Life Technologies) following the manufacturer's protocol. Each kit was tested at 3 different sites to assess reproducibility. Total RNA and ERCC RNA spike-in control mixes from the same lots were sent to 12 ABRF lab sites for amplification and cDNA generation. Ideally, this would have resulted in 36 different amplified samples, 3 from each input RNA. Libraries were constructed at one site from the amplified cDNAs using the TruSeq RNA library preparation kit on the Tecan Freedom EVO Liquid Handling Robot. As an unamplified control, ribosomal depletion and PolyA selection were performed separately using 5ng, 100ng and 1ug of total RNA prior to library construction. All libraries were pooled and sequenced using the Illumina HiSeq platform. An overview of the study and the results will be presented.

Nicolet, Charles; Paulson, Ariel; Shanker, Savita; Beckloff, N.; Bintzler, D.; Bivens, N. J.; Davis, R. R.; Donnelly, R. J.; Edenberg, H. J.; Gillaspy, A. F.; Grove, D.; Jafari, N.; Kerley-Hamilton, J. S.; Lashley, K.; Lyons, R. H.; Peak, A.; Perera, A.; Thimmapuram, J.; Wang, L.; Wright, C. L.; Alekseyev, Y.

2013-01-01

129

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes  

Microsoft Academic Search

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence.

David B. Stern; Tony P. Hodge; David M. Lonsdale

1984-01-01

130

Increased systemic oxidatively generated DNA and RNA damage in schizophrenia.  

PubMed

Schizophrenia is associated with a substantially increased somatic morbidity and mortality, which may partly be caused by accelerated cellular aging. Oxidative stress is an established mediator of aging and a suggested aetiological mechanism in both schizophrenia and age-related medical disorders such as cardiovascular disease, type 2 diabetes and dementia. We determined the urinary excretion of markers of systemic Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, respectively, in 40 schizophrenia patients and 40 age- and sex-matched controls, using ultra-performance liquid chromatography with tandem mass spectrometry. Measures of psychopathology, perceived stress and cortisol secretion were collected. Patients were re-examined after four months. We found a 20% increase in the median excretion of both markers in schizophrenia patients vs. healthy controls (P=0.003 and <0.001, respectively). This difference persisted after the adjustment for multiple demographical, lifestyle and metabolic factors. In patients, the marker excretion was not influenced by medication load, and was not driven by symptom severity, perceived stress or cortisol secretion, neither at baseline nor in relation to changes at follow-up. We conclude that schizophrenia is associated with increased systemic nucleic acid damage from oxidation, which could constitute a molecular link between schizophrenia and its associated signs of accelerated aging. PMID:23465294

Jorgensen, Anders; Broedbaek, Kasper; Fink-Jensen, Anders; Knorr, Ulla; Greisen Soendergaard, Mia; Henriksen, Trine; Weimann, Allan; Jepsen, Peter; Lykkesfeldt, Jens; Enghusen Poulsen, Henrik; Balslev Jorgensen, Martin

2013-03-01

131

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

PubMed Central

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

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

132

Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam.  

PubMed

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis. PMID:16328977

Kim, Sun-Hyung; Hamada, Tatsuro

2005-12-01

133

Advantageous sensitivity in the DNA homolog of the RNA dopamine aptamer.  

PubMed

A competitive enzyme-linked aptamer assay for DA was performed by using two aptamers, individually; one is a 57 mer-RNA aptamer and the other is its homolog DNA aptamer. The difference between the RNA aptamer and the DNA aptamer are based on their particular nucleotides. It is known that the lack of a hydroxyl group in the 2' position of DNA is related with its chemical and biological stability. Thus, the use of the DNA homolog of the RNA aptamer could improve the affinity toward DA due to stability and finally, lower the detection limit. In this paper, we report advantageous sensitivity and specificity of its homolog DNA aptamer assay as compared to the RNA aptamer assay. Both aptamer assays were performed with 0.01 µg mL(-1) of each aptamer and 1.205 × 10(-8) M DA-HRP conjugate using the optimized method. A dose-response curve was constructed, and the limit of detection for the DA was determined as 6.3 × 10(-8) M for RNA aptamer assay, and 3.2 × 10(-12) M for the homolog DNA aptamer assay, respectively. These results demonstrated that the assay sensitivity was more than 10(4) times improved with the DNA homolog of the RNA aptamer compared to its original RNA aptamer obtained through SELEX process. Also these results confirmed that the DNA homolog of the RNA aptamer can maintained the binding site and retained a function in both structure. Thus, the switching to the DNA version of RNA aptamer is possible to bind more stably and still able to bind to dopamine. PMID:24063619

Kim, Eunhye; Paeng, Insook Rhee

2014-01-01

134

Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp  

Microsoft Academic Search

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toaamanitin even up to a concentration of 250µg\\/ml.

Pranab K. Sadhukhan; Asit K. Chakraborty; Arindam Dasgupta; Hemanta K. Majumder

1997-01-01

135

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.

Montecucco, Alessandra; Biamonti, Giuseppe

2013-01-01

136

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

PubMed

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), mRNA, noncoding RNA (ncRNA; enriched in miRNA) and protein from the same tissues. After tissue selection, about 12-16 extractions of DNA, RNA or 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

Peńa-Llopis, Samuel; Brugarolas, James

2013-10-17

137

Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA  

PubMed Central

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the ? or ? globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants. Images

Poon, Raymond; Paddock, Gary V.; Heindell, Howard; Whitcome, Philip; Salser, Winston; Kacian, Dan; Bank, Arthur; Gambino, Roberto; Ramirez, Francesco

1974-01-01

138

Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing.  

PubMed Central

We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the RNA editing of transcripts encoding glutamate-gated ion channels in brain. Images

Kim, U; Wang, Y; Sanford, T; Zeng, Y; Nishikura, K

1994-01-01

139

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

2013-05-17

140

Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus.  

PubMed Central

RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm. Images

Dasmahapatra, B; Dasgupta, R; Saunders, K; Selling, B; Gallagher, T; Kaesberg, P

1986-01-01

141

Rapid Determination of RNA Accessible Sites by Surface Plasmon Resonance Detection of Hybridization to DNA arrays  

PubMed Central

RNA accessible sites are the regions in an RNA molecule, which are available for hybridization with complementary DNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of ~75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array comprised of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.

Mandir, Joshua B.; Lockett, Matthew R.; Phillips, Margaret F.; Allawi, Hatim T.; Lyamichev, Victor I.; Smith, Lloyd M.

2009-01-01

142

Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays.  

PubMed

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of approximately 75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner. PMID:19874056

Mandir, Joshua B; Lockett, Matthew R; Phillips, Margaret F; Allawi, Hatim T; Lyamichev, Victor I; Smith, Lloyd M

2009-11-01

143

Systematic comparison of the fidelity of aRNA, mRNA and TRNA on gene expression profiling using cDNA microarray  

Microsoft Academic Search

In cDNA microarray technology, there are three main reverse transcription based RNA labeling methods, using total RNA (T-RNA), mRNA, and amplified antisense RNA (aRNA), respectively. However, despite the common use of the three types of RNAs, limited data are available regarding their differences and concordances. In this report, we compared the three methods through two sets of self-comparison experiments using

Yao Li; Tao Li; Sanzhen Liu; Minyan Qiu; Zhiyong Han; Zhenling Jiang; Rongyu Li; Kang Ying; Yi Xie; Yumin Mao

2004-01-01

144

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

145

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

146

Modified method for combined DNA and RNA isolation from peanut and other oil seeds.  

PubMed

Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds. PMID:23104473

Dang, Phat M; Chen, Charles Y

2012-10-29

147

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.

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

2011-01-01

148

Interaction of arsenic trioxide As2O3 with DNA and RNA.  

PubMed

Arsenic salts have been used for centuries to treat a variety of medical conditions ranging from infectious disease to cancer. More recently, trivalent arsenic trioxide was found to exhibit high antitumor activity towards hematological malignancies. Even though much is known about antitumor activity and DNA damage by As2O3, there has been no report on the interaction of arsenic trioxide with isolated DNA or RNA. Therefore, it was of interest to examine the interaction of As2O3 with DNA and RNA in aqueous solution at physiological pH. FTIR and UV-visible difference spectroscopic methods were used to characterize the nature of drug-DNA and drug-RNA interactions and to determine the As binding site, the binding constant, the sequence selectivity, the helix stability, and the biopolymer secondary structure in the As2O3-polynucleotide complexes in vitro. The FTIR spectroscopic studies were conducted with As2O3-polynucleotide (phosphate) ratios of 1/40, 1/20, 1/10, and 1/5, with a final DNA (P) or RNA (P) concentration of 6.25 mmol/l. Spectroscopic results showed As2O3 binds to DNA and RNA at G-C, A-T, and A-U bases, and no interaction with the backbone PO2 group. As2O3-DNA and -RNA adducts showed one type of binding with overall binding constant of K(As2O3-DNA) = 1.24 x 10(5) M(-1) and K(As2O3-RNA) = 2.60 x 10(5) M(-1). The As2O3-polynucleotide complexation is associated with a partial biopolymer aggregation and no major alterations of B-DNA or A-RNA structure. PMID:16225394

Nafisi, Shohreh; Sobhanmanesh, Amir; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Tajmir-Riahi, Heidar-Ali

2005-10-01

149

A comparative study on the interaction of cis- and trans-platin with DNA and RNA.  

PubMed

Cis-diamminedichloroplatinum(II) is a frequently used and very effective chemotherapeutic drug for treatment of various malignancies; however, the trans isomer is clinically ineffective. Cis-platin exerts its antitumor activity by binding to DNA via intrastrand cross-links to d(GpG) (dG = deoxyguanosine) and to d(ApG) (dA = deoxyadenosine), interfering with DNA replication and transcription and causing cell death. The trans-diamminedichloroplatinum(II) isomer also binds DNA, but is clinically ineffective. This study was designed to examine the interactions of cis- and trans-platin with calf thymus DNA and yeast RNA in aqueous solution at physiological conditions, using a constant DNA and RNA concentration (6.25 mM) and various platin salts/polynucleotide (phosphate) ratios of 1/100, 1/50, 1/25, and 1/12.5. Fourier transform infrared, ultraviolet-visible spectroscopic methods were used to determine the drug binding modes, the binding constants, and the stability of cis- and trans-platin-DNA and -RNA complexes in aqueous solution. Spectroscopic evidence showed that cis- and trans-platin bind to the major and minor grooves of DNA (via G, A, T, and C bases), while RNA binding is through G, U, A, and C bases with some degree of the pt-phosphate (PO(2)) interaction for both isomers and overall binding constants of K((cis-platin-DNA)) = 5.51 x 10(4) M(-1), K((trans-platin-DNA)) = 2.26 x 10(4) M(-1), K((cis-platin-RNA)) = 1.9 x 10(4) M(-1), and K((trans-platin-RNA)) = 1.75 x 10(4) M(-1). DNA and RNA aggregations occurred at high platin concentrations. No biopolymer conformational changes were observed upon cis- and trans-platin interactions, while DNA remains in the B-family, and RNA retains its A-family structure. The order of platin compound-polymer stability was cis-platin-DNA > trans-platin-DNA > cis-platin-RNA > trans-platin-RNA. PMID:19558218

Nafisi, Shohreh; Norouzi, Zeinab

2009-09-01

150

siRNAs from miRNA sites mediate DNA methylation of target genes.  

PubMed

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants. PMID:20621980

Chellappan, Padmanabhan; Xia, Jing; Zhou, Xuefeng; Gao, Shang; Zhang, Xiaoming; Coutino, Gabriela; Vazquez, Franck; Zhang, Weixiong; Jin, Hailing

2010-07-09

151

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.

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

2008-01-01

152

Beyond RNA and DNA: In-Situ Sequencing of Informational Polymers  

NASA Astrophysics Data System (ADS)

Semiconductor sequencing and nascent nanopore sequencing enable in situ sequencing of RNA, DNA, and non-standard informational polymers. This can help bring high sensitivity and specificity to the search for life on Mars, Enceladus, and Europa.

Carr, C. E.; Ruvkun, G.; Zuber, M. T.

2012-10-01

153

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...

154

Evolution from DNA to RNA Recognition by the bI3 LAGLIDADG Maturase  

SciTech Connect

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 Angstroms from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.

Longo,A.; Leonard, C.; Bassi, G.; Berndt, D.; Krahn, J.; Tanaka Hall, T.; Weeks, K.

2005-01-01

155

Quantitative Studies of Phytohemagglutinin-Induced DNA and RNA Synthesis in Normal and Agammaglobulinemic Leukocytes.  

National Technical Information Service (NTIS)

Leukocytes from nine patients with acquired agammaglobulinemia were studied in vitro. Synthesis of deoxyribonucleic acid (DNA) and ribonculeic acid (RNA) induced by phytohemagglutinin was measured by determination of the degree of incorporation of labeled...

D. C. Tormey R. Komin H. H. Fudenberg

1967-01-01

156

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography  

PubMed Central

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community’s genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

Fadrosh, Douglas W.; Andrews-Pfannkoch, Cynthia; Williamson, Shannon J.

2011-01-01

157

Development of a Chimeric DNA-RNA Hammerhead Ribozyme Targeting SARS Virus  

Microsoft Academic Search

Objective: Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Method: Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To

Akiko Fukushima; Noboru Fukuda; Yimu Lai; Takahiro Ueno; Mitsuhiko Moriyama; Fumihiro Taguchi; Akifumi Iguchi; Kazushi Shimizu; Kazumichi Kuroda

2009-01-01

158

Tissue effect on RNA:DNA ratios of marine fish larvae  

Microsoft Academic Search

SUMMARY: In some routine studies of larval condition based on RNA:DNA ratios, heads and\\/or guts are removed for fur- ther age and feeding analysis. Also, during capture larvae are often found with their eyes missing. In this work we analysed tissues effects (muscle, head, eye, gut and the whole larvae) on RNA:DNA ratios from different species (Sardina pilchardus, Engraulis encrasicolus,

M. Pilar Olivar; Marina V. Diaz; M. Alexandra Chícharo

2009-01-01

159

Partial titin cDNA sequence isolated from rabbit cardiac muscle RNA  

Microsoft Academic Search

Two regions of the rabbit cardiac titin cDNA were amplified from rabbit cardiac muscle total RNA using primers based on rabbit skeletal muscle titin (connectin) cDNAs. These 1.7 kb and 1.5 kb RNA-PCR products were based on the 3' regions of the skeletal muscle titin clones CE12 and MS2, respectively. The cDNA sequence of the 1.7 kb product was extended

Jeffery D. Fritz; Jon A. Wolff; Marion L. Greaser

1993-01-01

160

Chimeric (aeg-pyrrolidine)PNAs: synthesis and stereo-discriminative duplex binding with DNA/RNA.  

PubMed

The design and facile conversion of naturally occurring 4-hydroxyproline to all four diastereomers of thymine pyrrolidine PNA monomer, (2R,4S)-adenine, -guanine and -cytosine monomers and their incorporation into duplex forming PNA oligomers is reported. The interesting results of the hybridization studies with complementary DNA/RNA sequences in either parallel or antiparallel orientation reveal the stereochemistry-dependent DNA vs. RNA discriminations and parallel/antiparallel orientation selectivity. PMID:15351824

Lonkar, Pallavi S; Ganesh, Krishna N; Kumar, Vaijayanti A

2004-08-17

161

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

NASA Astrophysics Data System (ADS)

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

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

162

Macromolecule synthesis in Escherichia coli BB under various growth conditions.  

PubMed Central

The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures as investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.

Chohji, T; Sawada, T; Kuno, S

1976-01-01

163

DNA-RNA bodies in midgut cells of the stick insect, Bacillus rossius.  

PubMed

In the anterior part of the midgut and in the Malpighian tubules of the stick insect Bacillus rossius, about 10% of the epithelial cells develop endonuclear bodies which appear as DNA-RNA masses; in these cells the usual nucleoli are no longer evident. The DNA-RNA bodies are first formed in third instar larvae, become numerous in the fourth instar and persist in adults. In all larval instars and adults a different kind of DNA-body has been noticed in the epithelial cells of the posterior midgut. The DNA-RNA bodies of the anterior midgut and of the Malpighian tubules have been interpreted as the result of somatic gene amplification, whereas the DNA masses of the posterior midgut are likely due to a virus infection. PMID:1176909

Scali, V; Montanelli, E

1975-09-01

164

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

165

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids  

PubMed Central

Selective 2?-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2?-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (Mr = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2?-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.

Turner, Kevin B.; Yi-Brunozzi, Hye Young; Brinson, Robert G.; Marino, John P.; Fabris, Daniele; Le Grice, Stuart F.J.

2009-01-01

166

Evidence for DNA Cleavage Caused Directly by a transfer RNA-Targeting Toxin.  

PubMed

The killer yeast species Pichiaacaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNA(Gln) mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation. PMID:24069426

Shigematsu, Megumi; Ogawa, Tetsuhiro; Tanaka, Wataru; Takahashi, Kazutoshi; Kitamoto, Hiroko K; Hidaka, Makoto; Masaki, Haruhiko

2013-09-17

167

Unusual sequences, homologous to 5S RNA, in ribosomal DNA repeats of the nematode Meloidogyne arenaria.  

PubMed

There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain "lower eukaryotes" (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes. Evidence is presented for rearrangements and deletions within Meloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences in Meloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families. PMID:3138424

Vahidi, H; Curran, J; Nelson, D W; Webster, J M; McClure, M A; Honda, B M

1988-01-01

168

A Third Recognition Element in Bacterial Promoters: DNA Binding by the alpha Subunit of RNA Polymerase  

Microsoft Academic Search

A DNA sequence rich in (A + T), located upstream of the -10, -35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP). When fused to other promoters, such

Wilma Ross; Khoosheh K. Gosink; Julia Salomon; Kazuhiko Igarashi; Chao Zou; Akira Ishihama; Konstantin Severinov; Richard L. Gourse

1993-01-01

169

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

PubMed

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

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

2012-08-08

170

RNase-Like Domain in DNA-Directed RNA Polymerase II  

Microsoft Academic Search

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared

Tsuyoshi Shirai; Mitiko Go

1991-01-01

171

Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation.  

PubMed

It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribed RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine. PMID:16143700

Mizuta, Ryushin; Mizuta, Midori; Kitamura, Daisuke

2005-09-05

172

Isolation of RNA, DNA, and Proteins from Formalin-Fixed Paraffin-Embedded Tissue Specimens.  

National Technical Information Service (NTIS)

Methods are disclosed for rapid, reliable and simple isolation of RNA, DNA and proteins from formalin-fixed paraffin-embedded tissue samples. RNA purified in this manner can be used to monitor gene expression levels. The tissue sample can be a tumor or ot...

K. D. Danenberg P. V. Daneberg S. Swenson

2006-01-01

173

Regulation of human RNA polymerase III transcription by DNMT1 and DNMT3a DNA methyltransferases.  

PubMed

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks. PMID:22219193

Selvakumar, Tharakeswari; Gjidoda, Alison; Hovde, Stacy L; Henry, R William

2012-01-04

174

Preservation of RNA and DNA from mammal samples under field conditions.  

PubMed

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months. PMID:23617785

Camacho-Sanchez, Miguel; Burraco, Pablo; Gomez-Mestre, Ivan; Leonard, Jennifer A

2013-04-26

175

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.  

PubMed

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing. PMID:22745249

Jinek, Martin; Chylinski, Krzysztof; Fonfara, Ines; Hauer, Michael; Doudna, Jennifer A; Charpentier, Emmanuelle

2012-06-28

176

RNA Polymerase Switch in Transcription of Yeast rDNA: Role of Transcription Factor UAF (Upstream Activation Factor) in Silencing rDNA Transcription by RNA Polymerase II  

Microsoft Academic Search

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without

Loan Vu; Imran Siddiqi; Bum-Soo Lee; Cathleen A. Josaitis; Masayasu Nomura

1999-01-01

177

[Optimization of T7-based RNA amplification system for cDNA microarray].  

PubMed

cDNA microarrays are powerful parallel tools for gene expression profiling analysis, which help us to understand the molecular mechanism of diseases and to identify potential targets for therapeutic intervention. However, their broader application are hampered by the large amount of RNA required: up to 200 microg of total RNA or 5 microg of mRNA for one chip, making analysis of small samples difficult. In this work, combined with a template switching effect, the T7 RNA linear amplification procedure was optimized, providing multiple copies of anti-sense RNA with reduced sample inputs: no more than 3 microg total RNA for one chip. Using the same RNA sample in all cases, the anti-sense RNA labeling method was compared with standard total RNA and mRNA methods by two sets of self-comparison experiments. Furthermore, the three methods in profiling analysis were compared with the same pair RNA samples. All the results indicated that the fidelity, reproducibility, and reliability showed no significant difference with conventional total RNA or mRNA microarrays. PMID:12766805

Li, Tao; Li, Yao; Han, Zhi-Yong; Chen, Qin; Qiu, Min-Yan; Ni, Sheng; Jiang, Zhen-Ling; Xie, Yi; Mao, Yu-Min

2003-05-01

178

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors  

Microsoft Academic Search

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5?-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the

M Ehrlich; C B Woods; M C Yu; L Dubeau; F Yang; M Campan; D J Weisenberger; Ti Long; B Youn; E S Fiala; P W Laird

2006-01-01

179

Two-Step Recruitment of RNA-Directed DNA Methylation to Tandem Repeats  

Microsoft Academic Search

Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence

Simon W.-L. Chan; Xiaoyu Zhang; Yana V. Bernatavichute; Steven E. Jacobsen

2006-01-01

180

Head-On Collision Between a DNA Replication Apparatus and RNA Polymerase Transcription Complex  

Microsoft Academic Search

An in vitro system reconstituted from purified proteins has been used to examine what happens when the DNA replication apparatus of bacteriophage T4 collides with an Escherichia coli RNA polymerase ternary transcription complex that is poised to move in the direction opposite to that of the moving replication fork. In the absence of a DNA helicase, the replication fork stalls

Bin Liu; Bruce M. Alberts

1995-01-01

181

In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae.  

PubMed Central

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.

Ishikawa, M; Janda, M; Krol, M A; Ahlquist, P

1997-01-01

182

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

PubMed Central

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

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

2012-01-01

183

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

2013-09-08

184

Affinity selection of DNA-binding protein complexes using mRNA display  

Microsoft Academic Search

Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro sel- ection of DNA-binding protein heterodimeric com- plexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultan- eously enriched about 100-fold

Seiji Tateyama; Kenichi Horisawa; Hideaki Takashima; Etsuko Miyamoto-Sato; Nobuhide Doi; Hiroshi Yanagawa

2006-01-01

185

Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor  

Microsoft Academic Search

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid\\/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is

Kilian Dill; Samuel D. H Chan; Thomas W Gibbs

1997-01-01

186

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA  

Microsoft Academic Search

Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb

Zhun Lu; Muthusamy Kunnimalaiyaan; Brent L. Nielsen

1996-01-01

187

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

188

Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level.  

PubMed

Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger. PMID:23214411

Herrero-Galán, Elías; Fuentes-Perez, Maria Eugenia; Carrasco, Carolina; Valpuesta, José M; Carrascosa, José L; Moreno-Herrero, Fernando; Arias-Gonzalez, J Ricardo

2012-12-24

189

The RNA Exosome Targets the AID Cytidine Deaminase to Both Strands of Transcribed Duplex DNA Substrates  

PubMed Central

SUMMARY Activation Induced cytidine Deaminase (AID) initiates Immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and non-template strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA processing/degradation complex, in targeting AID to both DNA strands. In B-lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for non-coding RNA surveillance machinery in generating antibody diversity.

Basu, Uttiya; Meng, Fei-Long; Keim, Celia; Grinstein, Veronika; Pefanis, Evangelos; Eccleston, Jennifer; Zhang, Tingting; Myers, Darienne; Wesemann, Duane R.; Januszyk, Kurt; Gregory, Richard I.; Deng, Haiteng; Lima, Christopher D.; Alt, Frederick W.

2011-01-01

190

Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis.  

PubMed Central

Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile). The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete. A majority of the tRNA genes are located in close proximity to one another. A unique feature of the Pro and Ile genes is that their DNA sequence overlap.

Desai, S M; Vaughan, J; Weiss, S B

1986-01-01

191

Quantitative PCR methods for RNA and DNA in marine sediments: maximizing yield while overcoming inhibition.  

PubMed

For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extraction kits and a bead-beating protocol with phenol/chloroform extraction steps. Purification methods included silica-column-based procedures from the MoBio kits, RNeasy MinElute, WizardPlus miniprep columns, and an acrylamide gel extraction. DNA and RNA purification with WizardPlus and RNeasy, respectively, led to significant losses of nucleic acids and archaeal 16S rRNA or 16S rRNA gene, as measured with RiboGreen or PicoGreen, and RT-qPCR or qPCR. Extraction and purification of DNA with the MoBio DNA UltraClean and DNA PowerSoil kits also decreased the yields slightly, relative to gel purification, in all sediments, except those from the deep sea in the Gulf of Mexico. Organic matter in humic-rich sediments may bind to these silica columns, reducing their nucleic acid-loading capacity. Purification with gel extraction cleans up organic-rich sediment samples sufficiently for quantitative analysis while avoiding the yield loss associated with commonly used silica columns. PMID:20059545

Lloyd, Karen G; Macgregor, Barbara J; Teske, Andreas

2009-12-07

192

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

2012-04-03

193

Evidence for DNA Cleavage Caused Directly by a transfer RNA-Targeting Toxin  

PubMed Central

The killer yeast species Pichiaacaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNAGlnmcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.

Shigematsu, Megumi; Ogawa, Tetsuhiro; Tanaka, Wataru; Takahashi, Kazutoshi; Kitamoto, Hiroko K.; Hidaka, Makoto; Masaki, Haruhiko

2013-01-01

194

Viral nanomotors for packaging of dsDNA and dsRNA  

PubMed Central

While capsid proteins are assembled around single-stranded genomic DNA or RNA in rod-shaped viruses, the lengthy double-stranded genome of other viruses is packaged forcefully within a preformed protein shell. This entropically unfavourable DNA or RNA packaging is accomplished by an ATP-driven viral nanomotor, which is mainly composed of two components, the oligomerized channel and the packaging enzymes. This intriguing DNA or RNA packaging process has provoked interest among virologists, bacteriologists, biochemists, biophysicists, chemists, structural biologists and computational scientists alike, especially those interested in nanotechnology, nanomedicine, AAA+ family proteins, energy conversion, cell membrane transport, DNA or RNA replication and antiviral therapy. This review mainly focuses on the motors of double-stranded DNA viruses, but double-stranded RNA viral motors are also discussed due to interesting similarities. The novel and ingenious configuration of these nanomotors has inspired the development of biomimetics for nanodevices. Advances in structural and functional studies have increased our understanding of the molecular basis of biological movement to the point where we can begin thinking about possible applications of the viral DNA packaging motor in nanotechnology and medical applications.

Guo, Peixuan; Lee, Tae Jin

2007-01-01

195

Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase.  

PubMed Central

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.

Auer, T; Sninsky, J J; Gelfand, D H; Myers, T W

1996-01-01

196

Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system  

PubMed Central

To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1 gm/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.

Luke, Jeremy; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

2009-01-01

197

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

198

Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription.  

PubMed Central

The poly(A)-containing mitochondrial mRNAs of rat liver were tested for their ability to serve as templates for the DNA synthesis by means of reverse transcription in the presence of the oligo(dT) primer and the RNA-directed DNA-polymerase from avian myeloblastosis virus. The mT-mRNA does not support the DNA synthesis in the standard conditions sufficient for effective reverse transcription of rabbit globin mRNA and of poly(A) in the presence of oligo(dT) primers. After a mild alkaline treatment of the mRNA and subsequent polyadenylation of the 3'-termini of the generated fragments with ATP:RNA adenyltransferase from E.coli the poly(A) (+) polyribonucleotides are able to serve as templates for reverse transcription in the presence of oligo(dT) and the reverse transcriptase. A conclusion is made that a "structural stop" exists in mitochondrial mRNA non-translable regions adjacent to the poly(A) terminal sequence. The "structural stop" is suggested to be caused by post-transcriptional modification of mRNA (methylation, etc.) or by a particularly stable secondary structure in this region of the mRNA molecules.

Frolova, L; Arsenyan, S; Avdonina, T; Gaitskhoki, V; Kisselev, O; Neifach, S; Kisselev, L

1978-01-01

199

Structural and Functional Consequences of Phosphate-Arsenate Substitutions in Selected Nucleotides: DNA, RNA, and ATP  

PubMed Central

A recent finding of a bacterial strain (GFAJ-1) that can rely on arsenic instead of phosphorus raised the questions of if and how arsenate can replace phosphate in biomolecules that are essential to sustain cell life. Apart from questions related to chemical stability, there are those of the structural and functional consequences of phosphate-arsenate substitutions in vital nucleotides in GFAJ1-like cells. In this study we selected three types of molecules (ATP/ADP as energy source and replication regulation; DNA–protein complexes for DNA replication and transcription initiation; and a tRNA–protein complex and ribosome for protein synthesis) to computationally probe if arsenate nucleotides can retain the structural and functional features of phosphate nucleotides. Hydrolysis of adenosine triarsenate provides 2–3 kcal/mol less energy than ATP hydrolysis. Arsenate DNA/RNA interacts with proteins slightly less strongly than phosphate DNA/RNA, mainly due to the weaker electrostatic interactions of arsenate. We observed that the weaker arsenate RNA–protein interactions may hamper rRNA assembly into a functional ribosome. We further compared the experimental EXAFS spectra of the arsenic bacteria with theoretical EXAFS spectra for arsenate DNA and rRNA. Our results demonstrate that while it is possible that dried GFAJ-1 cells contain linear arsenate DNA, the arsenate 70S ribosome does not contribute to the main arsenate depository in the GFAJ-1 cell. Our study indicates that evolution has optimized the inter-relationship between proteins and DNA/RNA, which requires overall changes at the molecular and systems biology levels when replacing phosphate by arsenate.

2012-01-01

200

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

PubMed Central

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

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

2012-01-01

201

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR  

PubMed Central

Background Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. Results We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65°C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. Conclusion Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

Del Aguila, Eduardo M; Dutra, Marcio B; Silva, Joab T; Paschoalin, Vania MF

2005-01-01

202

Characterization of the yeast tRNA Ser genomic organization and DNA sequence.  

PubMed Central

Purified, isolated yeast tRNA Ser2 was used as a hybridization probe to estimate the number of tRNA Ser2 genes in the yeast genome. Molecular clones of several of the genes were obtained. Three examples were studied in detail with respect to their genomic organization, and DNA sequences were determined for them. There appear to be eleven tRNA Ser2 genes in the yeast genome. They are neither tandemly repeated, nor clustered with other tRNA genes. They contain no intervening sequences. Images

Page, G S; Hall, B D

1981-01-01

203

Moving interfaces in rod-like macromolecules  

NASA Astrophysics Data System (ADS)

We present a model that describes mechanical unfolding behavior in rod-like macromolecules. We propose that the unfolding occurs via the motion of a folded/unfolded interface along the molecule. We predict the speed of this interface as a function of the pulling velocity such that the resulting force-extension curve replicates the overstretching transition typical of coiled coils and DNA. We model the molecules as one-dimensional continua capable of existing in two metastable states under an applied tension. The interface separates these two metastable states and represents a jump in stretch, which is related to the applied force by the worm-like chain relation. The Abeyaratne-Knowles theory of phase transitions in continua governs the mechanics of the interface.

Raj, Ritwik; Purohit, Prashant K.

2010-07-01

204

In vivo DNA/RNA adduction of 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BaP) in the liver of rainbow trout (Oncorhynchus mykiss)  

SciTech Connect

Juvenile rainbow trout were exposed via two intramuscular injections to either 14C-DMBA for 24 hr or 14C-BaP for 48 hr, after which the livers were removed for DNA extraction and analysis. In the fish exposed to 14C-BaP, 0.2 ng was bound to the DNA, representing 0.5% of the total liver PAH-derived radioactivity and 2.38% of the administered dose. Liver DNA and RNA were found to contain 0.5% of the administered dose, respectively. Liver analysis of rainbow trout exposed to 14C-DMBA demonstrated that 0.4 ng and 0.3 ng were bound to the DNA and RNA, respectively. This represents 1.0% and 0.6% of the liver DMBA burden, respectively. The DNA adduct concentrations formed were comparable to both in vitro and in vivo experiments with both mammals and fishes, indicating that relatively small, environmentally realistic' doses of PAH have the ability to bind significantly to critical cellular macromolecules of young fish in vivo.

Schnitz, A.R.; O'Connor, J.M. (New York University Medical Center, NY (United States))

1992-07-01

205

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

PubMed

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(2+) at the active site. A subset of amino acids engaged in contacts to RNA 2'-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. PMID:18719385

Pallan, Pradeep S; Egli, Martin

2008-08-18

206

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

207

DNA-Directed Expression of Functional Flock House Virus RNA1 Derivatives in Saccharomyces cerevisiae, Heterologous Gene Expression, and Selective Effects on Subgenomic mRNA Synthesis  

PubMed Central

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3?-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.

Price, B. Duane; Roeder, Mark; Ahlquist, Paul

2000-01-01

208

MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression  

PubMed Central

Precise regulation of DNA damage response is crucial for cellular survival after DNA damage, and its abrogation often results in genomic instability in cancer. Phosphorylated histone H2AX (?H2AX) forms nuclear foci at sites of DNA damage and facilitates DNA damage response and repair. MicroRNAs are short, non-protein-encoding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation of and/or degrading mRNA. How microRNAs modulate DNA damage response is largely unknown. In this study, we developed a cell-based screening assay utilizing ionizing radiation-induced ?H2AX foci formation in a human osteosarcoma cell line, U2OS, as the readout. By screening a library of human microRNA mimics, we identified several microRNAs that inhibited ?H2AX foci formation. Among them, miR-138 directly targeted the histone H2AX 3?-UTR, reduced histone H2AX expression and induced chromosomal instability after DNA damage. Overexpression of miR-138 inhibited homologous recombination and enhanced cellular sensitivity to multiple DNA damaging agents (cisplatin, camptothecin, and ionizing radiation). Reintroduction of histone H2AX in miR-138 overexpressing cells attenuated miR-138-mediated sensitization to cisplatin and camptothecin. Our study suggests that miR-138 is an important regulator of genomic stability and a potential therapeutic agent to improve the efficacy of radiotherapy and chemotherapy with DNA damaging agents.

Wang, Yemin; Huang, Jen-Wei; Li, Ming; Cavenee, Webster K.; Mitchell, Patrick S.; Zhou, Xiaofeng; Tewari, Muneesh; Furnari, Frank B.; Taniguchi, Toshiyasu

2011-01-01

209

Enhanced specificity of HPV16 E6E7 siRNA by RNA–DNA chimera modification  

Microsoft Academic Search

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1–8 and its complementary part of the passenger

K Yamato; N Egawa; S Endo; K Ui-Tei; T Yamada; K Saigo; I Hyodo; T Kiyono; I Nakagawa

2011-01-01

210

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM  

PubMed Central

By dissecting and reconstituting a cell-free influenza virus genome replication system, we have purified and identified the minichromosome maintenance (MCM) complex, which is thought to be a DNA replicative helicase, as one of the host factors that regulate the virus genome replication. MCM interacted with the PA subunit of the viral RNA-dependent RNA polymerase that is found to be involved in the replication genetically. The virus genome replication was decreased in MCM2 knockdown cells. The viral polymerase appeared to be a nonproductive complex, that is, it was capable of initiating replication but produced only abortive short RNA chains. MCM stimulated de novo-initiated replication reaction by stabilizing a replication complex during its transition from initiation to elongation. Based on the findings, including the result that the MCM-mediated RNA replication reaction was competed with exogenously added RNA, we propose that MCM functions as a scaffold between the nascent RNA chains and the viral polymerase.

Kawaguchi, Atsushi; Nagata, Kyosuke

2007-01-01

211

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.  

PubMed

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation. PMID:16532039

Ehrlich, M; Woods, C B; Yu, M C; Dubeau, L; Yang, F; Campan, M; Weisenberger, D J; Long, Ti; Youn, B; Fiala, E S; Laird, P W

2006-04-27

212

Structural probing of the HIV1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands  

Microsoft Academic Search

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not

Kevin B. Turner; Robert G. Brinson; Hye Young Yi-Brunozzi; Jason W. Rausch; Jennifer T. Miller; S. F. J. Le Grice; John P. Marino; Daniele Fabris

2008-01-01

213

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

PubMed Central

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

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

2000-01-01

214

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

PubMed

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 G(1370) of human 16 S rRNA. PMID:24036117

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

2013-09-13

215

High-quality RNA preparation from Rhodosporidium toruloides and cDNA library construction therewith.  

PubMed

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of ?-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 ?g total RNA per gram of cells was obtained with an average purity measured as A???/A??? of 2.09. A titer of 10? cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation. PMID:20721646

Yang, Fan; Tan, Haidong; Zhou, Yongjin; Lin, Xinping; Zhang, Sufang

2011-02-01

216

Quantitation of cell-free DNA and RNA in plasma during tumor progression in rats  

PubMed Central

Background To clarify the implications of cell-free nucleic acids (cfNA) in the plasma in neoplastic disease, it is necessary to determine the kinetics of their release into the circulation. Methods To quantify non-tumor and tumor DNA and RNA in the plasma of tumor-bearing rats and to correlate such levels with tumor progression, we injected DHD/K12-PROb colon cancer cells subcutaneously into syngenic BD-IX rats. Rats were sacrificed and their plasma was analyzed from the first to the eleventh week after inoculation. Results The release of large amounts of non-tumor DNA into plasma was related to tumor development from its early stages. Tumor-specific DNA was detected in 33% of tumor-bearing rats, starting from the first week after inoculation and at an increasing frequency thereafter. Animals that were positive for tumor DNA in the plasma had larger tumors than those that were negative (p?=?0.0006). However, the appearance of both mutated and non-mutated DNA fluctuated with time and levels of both were scattered among individuals in each group. The release of non-tumor mRNA was unaffected by tumor progression and we did not detect mutated RNA sequences in any animals. Conclusions The release of normal and tumor cfDNA into plasma appeared to be related to individual-specific factors. The contribution of tumor DNA to the elevated levels of plasma DNA was intermittent. The release of RNA into plasma during cancer progression appeared to be an even more selective and elusive phenomenon than that of DNA.

2013-01-01

217

DNA Vector-based RNA Interference to Study Gene Function in Cancer  

PubMed Central

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

Stovall, Daniel B.; Wan, Meimei; Zhang, Qiang; Dubey, Purnima; Sui, Guangchao

2012-01-01

218

DNA vector-based RNA interference to study gene function in cancer.  

PubMed

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution. PMID:22710444

Stovall, Daniel B; Wan, Meimei; Zhang, Qiang; Dubey, Purnima; Sui, Guangchao

2012-06-04

219

Protein interactions with piALU RNA indicates putative participation of retroRNA in the cell cycle, DNA repair and chromatin assembly  

PubMed Central

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.

Blackwell, Benjamin J.; Lopez, Mary F.; Wang, Jianrong; Krastins, Bryan; Sarracino, David; Tollervey, James R.; Dobke, Marek; Jordan, I. King; Lunyak, Victoria V.

2012-01-01

220

Subunit Compositions of Arabidopsis DNA-Dependent RNA Polymerases and the Roles of the Plant-Specific RNA Polymerases IV and V in Gene Silencing  

Microsoft Academic Search

In addition to RNA polymerases I, II and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play non-redundant roles in siRNA-directed DNA methylation and gene silencing. Using a combination of affinity purification and protein identification by mass spectrometry, my studies show that Arabidopsis Pol

Thomas Ream

2009-01-01

221

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis  

Microsoft Academic Search

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase III. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number

Ming Li; Baerbel Rohrer

2006-01-01

222

Rapid Switching of TFIIH between RNA Polymerase I and II Transcription and DNA Repair In Vivo  

Microsoft Academic Search

The transcription\\/repair factor TFIIH operates as a DNA helix opener in RNA polymerase II (RNAP2) transcription and nucleotide excision repair. To study TFIIH in vivo, we generated cell lines expressing functional GFP-tagged TFIIH. TFIIH was homogeneously distributed throughout the nucleus with nucleolar accumulations. We provide in vivo evidence for involvement of TFIIH in RNA polymerase I (RNAP1) transcription. Photobleaching revealed

Deborah Hoogstraten; Alex L Nigg; Helen Heath; Leon H. F Mullenders; Roel van Driel; Jan H. J Hoeijmakers; Wim Vermeulen; Adriaan B Houtsmuller

2002-01-01

223

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

Microsoft Academic Search

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

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

2006-01-01

224

Flow cytometric detection and enumeration of DNA and RNA viruses infecting marine eukaryotic microalgae  

Microsoft Academic Search

Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large\\u000a DNA algal viruses HaV and HcV

Yuji Tomaru; Keizo Nagasaki

2007-01-01

225

[The mechanism of the proaggregant action on the thrombocytes of RNA and DNA molecules].  

PubMed

The entrance of extracellular Ca2+ in ADP-stimulated human platelets and phospholipase A2 activity have been studied under the action of Ca precipitates DNA, RNA and dsRNA. The nucleic acids and its Ca complexes (Ca-NA) have been found to induce the aggregation of isolated human platelets by stimulation of extracellular Ca2+ entrance into the platelets and the formation of phosphatidilcholine and phosphatidilethanolamine lysoderivatives having the membranolytic effect. PMID:1693862

Zakharian, R A; Rukhkian, L A

1990-01-01

226

Genome-wide evolutionary analysis of the noncoding RNA genes and noncoding DNA of Paramecium tetraurelia  

PubMed Central

The compact genome of the unicellular eukaryote Paramecium tetraurelia contains noncoding DNA (ncDNA) distributed into >39,000 intergenic sequences and >90,000 introns of 390 base pairs (bp) and 25 bp on average, respectively. Here we analyzed the molecular features of the ncRNA genes, introns, and intergenic sequences of this genome. We mainly used computational programs and comparative genomics possible because the P. tetraurelia genome had formed throughout whole-genome duplications (WGDs). We characterized 417 5S rRNA, snRNA, snoRNA, SRP RNA, and tRNA putative genes, 415 of which map within intergenic sequences, and two, within introns. The evolution of these ncRNA genes appears to have mainly involved purifying selection and gene deletion. We then compared the introns that interrupt the protein-coding gene duplicates arisen from the recent WGD and identified a population of a few thousands of introns having evolved under most stringent constraints (>95% of identity). We also showed that low nucleotide substitution levels characterize the 50 and 80–115 base pairs flanking, respectively, the stop and start codons of the protein-coding genes. Lower substitution levels mark the base pairs flanking the highly transcribed genes, or the start codons of the genes of the sets with a high number of WGD-related sequences. Finally, adjacent to protein-coding genes, we characterized 32 DNA motifs able to encode stable and evolutionary conserved RNA secondary structures and defining putative expression controlling elements. Fourteen DNA motifs with similar properties map distant from protein-coding genes and may encode regulatory ncRNAs.

Chen, Chun-Long; Zhou, Hui; Liao, Jian-You; Qu, Liang-Hu; Amar, Laurence

2009-01-01

227

Identification of recombinant plasmids containing DNA sequences derived from the 3? end of ovine thyroglobulin mRNA  

Microsoft Academic Search

This paper describes the cloning inE. coli of several DNA copies of the 3' portion of ovine thyroglobulin mRNA. The cDNA clones were identified byin situ cloning hybridization to32P-labelled thyroglobulin cDNA and by positive hybridization-translation assays. The longest thyroglobulin cDNA fragment cloned (psTg21A, 1670 bp), was shown by S1 nuclease mapping to be an unaltered copy of the thyroglobulin mRNA.

J. Chebath; Z. T. Tosta; O. Chabaud; M. Perricaudet

1982-01-01

228

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.

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

2013-01-01

229

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

230

DNA structure in human RNA polymerase II promoters1  

Microsoft Academic Search

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

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

1998-01-01

231

Human catalytic RNA and DNA-hydrolyzing antibodies  

Microsoft Academic Search

In patients with autoimmune diseases, anti-idiotypic antibodies directed to nucleoprotein complexes, DNA, and enzymes that participate in nucleic acid metabolism may be induced spontaneously by primary antigens and can have characteristics of the primary antigen, including catalytic activity. The first natural catalytic antibody, now termed abzyme, which hydrolyzes intestinal vasoactive peptide, was discovered by Paul et al. [Science 244 (1989)

Georgy A Nevinsky; Valentina N Buneva

2002-01-01

232

Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays  

PubMed Central

We have developed methods for using DNA array technology to probe the entire transcriptome to determine the RNA-binding specificity of ligands. Two methods were investigated. In the first method, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal was compared with a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect that the results will lead to the discovery of new aminoglycoside-binding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.

Liang, Fu-Sen; Greenberg, William A.; Hammond, Jennifer A.; Hoffmann, Julia; Head, Steven R.; Wong, Chi-Huey

2006-01-01

233

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase  

NASA Astrophysics Data System (ADS)

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

2001-01-01

234

'Knock down' of DNA polymerase beta by RNA interference: recapitulation of null phenotype.  

PubMed

DNA polymerase beta (pol beta) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol beta. Pol beta null (-/-) mouse embryos are not viable and pol beta null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol beta protein and mRNA to undetectable levels. Pol beta knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol beta null cells. Generation of pol beta knock down cells makes it possible to combine the pol beta null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol beta and its interactions with other proteins in mammalian cells. PMID:15380102

Polosina, Yaroslava Y; Rosenquist, Thomas A; Grollman, Arthur P; Miller, Holly

2004-11-01

235

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.  

PubMed Central

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.

Begu, D; Graves, P V; Domec, C; Arselin, G; Litvak, S; Araya, A

1990-01-01

236

The DNA strand of chimeric RNA\\/DNA oligonucleotides can direct gene repair\\/conversion activity in mammalian and plant cell-free extracts  

Microsoft Academic Search

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the

Howard B. Gamper; Hetal Parekh; Michael C. Rice; Michael Bruner; Heather Youkey; Eric B. Kmiec

2000-01-01

237

Spliced human immunodeficiency virus type 1 RNA is reverse transcribed into cDNA within infected cells.  

PubMed

Both the full-length and spliced RNA species of HIV-1 possess the necessary cis-acting elements including the primer binding site (PBS), the polypurine tract (PPT), as well as the 5' R and 3' R regions that are needed for their conversion to double-stranded cDNA through reverse transcription. Since measurable amounts of spliced viral RNA molecules can be detected within virus particles, we have examined the potential for reverse transcription of such virion-associated spliced viral RNA upon infection of permissive cells. Analysis of viral cDNA species by PCR and DNA sequencing not only led to the identification of viral DNA molecules that were reverse transcribed from full-length viral RNA, but also DNA molecules that displayed the same nucleotide sequences as those found in spliced viral RNA, except that the former harbored the complete long terminal repeats (LTR), a feature that distinguishes proviral DNA from viral genomic RNA. Further studies revealed various types of cDNA species that resemble the spliced viral RNA encoding each of the env, tat, rev, or nef genes, of which the nef cDNA represents the majority. Therefore, spliced HIV-1 RNA molecules must have been reverse transcribed along with full-length viral RNA during infection. PMID:15018708

Liang, Chen; Hu, Jing; Russell, Rodney S; Kameoka, Masanori; Wainberg, Mark A

2004-02-01

238

DNA Damage Increases the Levels of MDM2 Messenger RNA in wtp53 Human Cells  

Microsoft Academic Search

Damage to chromosomal DNA increases the levels of the transcriptional regulatory protein p53. We have investigated how the MDM2 protein, which binds to p53 and inactivates its transcriptional activity, may be controlled following DNA damage. Irradiation of human GM2149 fibro- blast cells causes an increase in MDM2 mRNA levels within 1 h, and levels remain elevated for at least 8

Brendan D. Price; Sonia J. Park

1994-01-01

239

Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II  

Microsoft Academic Search

THE RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB\\/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome2,3, suggests a functional link between transcription and DNA repair4,5. To understand the connection between these two cellular processes,

Ronny Drapkin; Joyce T. Reardon; Athar Ansari; Juch-Chin Huang; Leigh Zawel; Kyujeong Ahn; Aziz Sancar; Danny Reinberg

1994-01-01

240

HIV1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages  

Microsoft Academic Search

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Qß, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on

Ruth Levitz; Karl Drlica; Ellen Murphy

1994-01-01

241

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

242

Laser crosslinking of E. coli RNA polymerase and T7 DNA.  

PubMed Central

The first photochemical crosslinking of a protein to a nucleic acid using laser excitation is reported. A single, 120 mJ, 20 ns pulse at 248 nm crosslinks about 10% of bound E. coli RNA polymerase to T7 DNA under the conditions studied. The crosslinking yield depends on mercaptoethanol concentration, and is a linear function of laser intensity. The protein subunits crosslinked to DNA are beta, beta' and sigma.

Harrison, C A; Turner, D H; Hinkle, D C

1982-01-01

243

[Fibronectin fragmentation unmasks the activity stimulating DNA and RNA biosynthesis in granulation tissue cells in vitro].  

PubMed

Human blood plasma fibronectin decreased slightly the incorporation of precursors into nucleic acids of granulation tissue culture cells. A slight fragmentation of fibronectin, where the fragments with 180-200 kD molecular mass were developed, led to occurrence of the activity 2-fold stimulating the DNA synthesis. After more effective proteolysis using plasmin and trypsin the stimulating effect of fibronectin fragments on synthesis of nucleic acids maintained and constituted 165 +/- 12% and 127 +/- 7% for DNA and RNA, respectively. PMID:2437701

Zlatopol'ski?, A D; Za?denberg, M A; Berman, A E; Mazurov, V I; Karelin, A A

244

Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus  

Microsoft Academic Search

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently

MARK N. PRICHARD; SANJU JAIRATH; MARK E. T. PENFOLD; STEPHEN S. T. JEOR; MARLENE C. BOHLMAN; GREGORY S. PARI

245

Multiple protein\\/protein and protein\\/RNA interactions suggest roles for yeast DNA\\/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing  

Microsoft Academic Search

Sen1p in Saccharomyces cerevisiae is a Type I DNA\\/ RNA helicase. Mutations in the helicase domain per- turb accumulation of diverse RNA classes, and Sen1p has been implicated in 3˘ end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were

Doris Ursic; Karen Chinchilla; Jonathan S. Finkel; Michael R. Culbertson

2004-01-01

246

Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing  

PubMed Central

The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.

Hafner, Markus; Renwick, Neil; Farazi, Thalia A.; Mihailovi, Aleksandra; Pena, John T.G.; Tuschl, Thomas

2012-01-01

247

CTAB-urea method purifies RNA from melanin for cDNA microarray analysis.  

PubMed

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB)-urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB-urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results. PMID:15140079

Lagonigro, M Stefania; De Cecco, Loris; Carninci, Piero; Di Stasi, Delia; Ranzani, Tiziana; Rodolfo, Monica; Gariboldi, Manuela

2004-06-01

248

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

PubMed Central

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

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

2006-01-01

249

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

PubMed Central

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

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

2011-01-01

250

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms.  

PubMed

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured. PMID:16545766

De Mey, Marjan; Lequeux, Gaspard; Maertens, Jo; De Maeseneire, Sofie; Soetaert, Wim; Vandamme, Erick

2006-03-02

251

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; Espańola, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E. (UCLA); (MSKCC); (Delaware)

2012-10-23

252

Diffusion of macromolecules on lipid vesicles  

NASA Astrophysics Data System (ADS)

Diffusion of macromolecules on a surface of lipid vesicles of different reduced volume and geometry is investigated. It is assumed that the macromolecules deform the surface of the vesicles by inducing the spontaneous curvature which is proportional to their concentration. We study how nonuniform distribution of macromolecules is reflected in the shape of the vesicles and how the shape of the vesicles influences the diffusion process.

Gó?d?, W. T.

2009-01-01

253

Unspecific Cooperative Ligand Binding to One-Dimensional Lattice-like Macromolecules  

NASA Astrophysics Data System (ADS)

Unspecific ligand binding to one-dimensional lattice-like macromolecules, a common event in nature (e.g. ligand/protein binding to DNA and certain carbohydrates), presents distinctive features when compared to specific ligand binding to macromolecules. McGhee and von Hippel developed a mathematical formalism in the form of the Scatchard representation. This article presents the application of the theory for unspecific cooperative ligand binding to linear lattice-like macromolecules in isothermal titration calorimetry following an exact and accurate method, without the limitations and deficiencies of the Scatchard formalism.

Velazquez-Campoy, Adrian

2006-08-01

254

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.  

PubMed Central

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions. Images Figure 1 Figure 2 Figure 3

Tbakhi, A.; Totos, G.; Hauser-Kronberger, C.; Pettay, J.; Baunoch, D.; Hacker, G. W.; Tubbs, R. R.

1998-01-01

255

Role of Polyamines in Regulation of Sequence-Specific DNA Binding Activity  

Microsoft Academic Search

Polyamines are positively charged under physiological ionic and pH conditions. Therefore, the negatively charged macromolecules\\u000a in the cell, DNA, RNA, and certain proteins are natural targets for their interaction. Polyamine interaction with DNA was\\u000a considered to be electrostatic in nature and was theoretically interpreted in terms of the counterion condensation theory.\\u000a Early studies suggested stabilization of duplex DNA by natural

Sripriya Venkiteswaran; Thresia Thomas; T. J. Thomas

256

Quantitative PCR analysis of blood- and saliva-specific microRNA markers following solid-phase DNA extraction.  

PubMed

The use of mRNA for the identification of body fluids is of particular interest in forensic science, and increasing support has been demonstrated for the use of microRNA (miRNA) analysis. MiRNA is more stable than mRNA and has been shown to be differentially expressed in body fluids. No studies involving miRNA analysis from previously extracted DNA samples have yet been reported. The aim of this experiment was to determine if it was possible to conduct miRNA analysis on samples that were previously extracted using standard DNA extraction. Blood and saliva samples were extracted using DNA and RNA kits, followed by cDNA synthesis, and then underwent quantitative PCR analysis. A direct comparison of ?Ct values shows a larger abundance of miRNA following DNA extraction as opposed to total RNA extraction for both blood- and saliva-specific markers. By carrying out a comparison between the amounts of said markers, it could be seen that the expression of the blood-specific marker was higher in blood than in saliva, and vice versa for the saliva-specific marker. The results obtained could have a profound impact on cases for which the sample has already undergone DNA extraction, such as in cold cases. PMID:23333269

Omelia, Emma J; Uchimoto, Mari L; Williams, Graham

2013-01-16

257

Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity  

Microsoft Academic Search

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production

Fusao Takusagawa; Ken T. Takusagawa; Robert G. Carlson; Robert F. Weaver

1997-01-01

258

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.  

PubMed Central

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed. Images

Shih, D S; Park, I W; Evans, C L; Jaynes, J M; Palmenberg, A C

1987-01-01

259

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.

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

2013-01-01

260

Preparation of DNA and RNA Fragments Containing Guanine N2-Thioalkyl Tethers  

PubMed Central

This unit describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert-butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site-specific crosslinking to a thiol of a protein or another nucleic acid.

Hou, Xiaorong; Wang, Gang; Gaffney, Barbara L.; Jones, Roger A.

2010-01-01

261

Hydrogen sulfide induces oxidative damage to RNA and DNA in a sulfide-tolerant marine invertebrate.  

PubMed

Hydrogen sulfide acts as an environmental toxin across a range of concentrations and as a cellular signaling molecule at very low concentrations. Despite its toxicity, many animals, including the mudflat polychaete Glycera dibranchiata, are periodically or continuously exposed to sulfide in their environment. We tested the hypothesis that a broad range of ecologically relevant sulfide concentrations induces oxidative stress and oxidative damage to RNA and DNA in G. dibranchiata. Coelomocytes exposed in vitro to sulfide (0-3 mmol L(-1) for 1 h) showed dose-dependent increases in oxidative stress (as 2',7'-dichlorofluorescein fluorescence) and superoxide production (as dihydroethidine fluorescence). Coelomocytes exposed in vitro to sulfide (up to 0.73 mmol L(-1) for 2 h) also acquired increased oxidative damage to RNA (detected as 8-oxo-7,8-dihydroguanosine) and DNA (detected as 8-oxo-7,8-dihydro-2'-deoxyguanosine). Worms exposed in vivo to sulfide (0-10 mmol L(-1) for 24 h) acquired elevated oxidative damage to RNA and DNA in both coelomocytes and body wall tissue. While the consequences of RNA and DNA oxidative damage are poorly understood, oxidatively damaged deoxyguanosine bases preferentially bind thymine, causing G-T transversions and potentially causing heritable point mutations. This suggests that sulfide can be an environmental mutagen in sulfide-tolerant invertebrates. PMID:19327040

Joyner-Matos, Joanna; Predmore, Benjamin L; Stein, Jenny R; Leeuwenburgh, Christiaan; Julian, David

262

Role of messenger RNA-ribosome complex in complementary DNA display.  

PubMed

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (?10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. PMID:23558165

Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

2013-04-01

263

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

264

Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.  

PubMed

A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

2011-02-17

265

Maze training alters brain weights and cortical RNA\\/DNA ratios  

Microsoft Academic Search

In order to test whether training leads to anatomical and chemical changes in the brain, individual rats were given self-paced trials in mazes, traversing the maze in order to get from a food station to a water station. In 30 days of this training, during which they had no social interaction, the rats developed significant increases in weight and RNA\\/DNA

E. L. Bennett; M. R. Rosenzweig; H. Morimoto; M. Hebert

1979-01-01

266

Individual Basepair Stability of DNA and RNA Studied by NMR-Detected Solvent Exchange  

PubMed Central

In this study, we have optimized NMR methodology to determine the thermodynamic parameters of basepair opening in DNA and RNA duplexes by characterizing the temperature dependence of imino proton exchange rates of individual basepairs. Contributions of the nuclear Overhauser effect to exchange rates measured with inversion recovery experiments are quantified, and the influence of intrinsic and external catalysis exchange mechanisms on the imino proton exchange rates is analyzed. Basepairs in DNA and RNA have an approximately equal stability, and the enthalpy and entropy values of their basepair dissociation are correlated linearly. Furthermore, the compensation temperature, Tc, which is derived from the slope of the correlation, coincides with the melting temperature, and duplex unfolding occurs at that temperature where all basepairs are equally thermodynamically stable. The impact of protium-deuterium exchange of the imino hydrogen on the free energy of RNA basepair opening is investigated, and it is found that two A·U basepairs show distinct fractionation factors.

Steinert, Hannah S.; Rinnenthal, Jorg; Schwalbe, Harald

2012-01-01

267

Individual basepair stability of DNA and RNA studied by NMR-detected solvent exchange.  

PubMed

In this study, we have optimized NMR methodology to determine the thermodynamic parameters of basepair opening in DNA and RNA duplexes by characterizing the temperature dependence of imino proton exchange rates of individual basepairs. Contributions of the nuclear Overhauser effect to exchange rates measured with inversion recovery experiments are quantified, and the influence of intrinsic and external catalysis exchange mechanisms on the imino proton exchange rates is analyzed. Basepairs in DNA and RNA have an approximately equal stability, and the enthalpy and entropy values of their basepair dissociation are correlated linearly. Furthermore, the compensation temperature, T(c), which is derived from the slope of the correlation, coincides with the melting temperature, and duplex unfolding occurs at that temperature where all basepairs are equally thermodynamically stable. The impact of protium-deuterium exchange of the imino hydrogen on the free energy of RNA basepair opening is investigated, and it is found that two A·U basepairs show distinct fractionation factors. PMID:22713572

Steinert, Hannah S; Rinnenthal, Jörg; Schwalbe, Harald

2012-06-05

268

Mutation of Host dnaJ Homolog Inhibits Brome Mosaic Virus Negative-Strand RNA Synthesis  

PubMed Central

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.

Tomita, Yuriko; Mizuno, Tomomitsu; Diez, Juana; Naito, Satoshi; Ahlquist, Paul; Ishikawa, Masayuki

2003-01-01

269

Soaking of DNA into crystals of archaeal RNA polymerase achieved by desalting in droplets.  

PubMed

Transcription is a fundamental process across the three domains of life and is carried out by multi-subunit enzymatic DNA-directed RNA polymerases (RNAPs). The interaction of RNAP with nucleic acids is tightly controlled for precise and processive RNA synthesis. Whilst a wealth of structural information has been gathered on the eukaryotic Pol II in complex with DNA/RNA, no information exists on its ancestral counterpart archaeal RNAP. Thus, in order to extend knowledge of the archaeal transcriptional apparatus, crystallization of Sulfolobus shibatae RNAP (molecular mass of ~400 kDa) with DNA fragments was pursued. To achieve this goal, crystal growth was first optimized using a nanoseeding technique. An ad hoc soaking protocol was then put into place, which consisted of gently exchanging the high-salt buffer used for apo-RNAP crystal growth into a low-salt buffer necessary for DNA binding to RNAP. Of the various crystals screened, one diffracted to 4.3 Ĺ resolution and structural analysis showed the presence of bound DNA [Wojtas et al. (2012). Nucleic Acids Res. 40, doi:10.1093/nar/gks692]. PMID:22949213

Wojtas, Magdalena N; Abrescia, Nicola G A

2012-08-31

270

Single-molecule imaging of RNA polymerase-DNA interactions in real time.  

PubMed Central

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

Harada, Y; Funatsu, T; Murakami, K; Nonoyama, Y; Ishihama, A; Yanagida, T

1999-01-01

271

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

PubMed Central

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.

Chene, Arnaud; Vembar, Shruthi S.; Riviere, Loic; Lopez-Rubio, Jose Juan; Claes, Aurelie; Siegel, T. Nicolai; Sakamoto, Hiroshi; Scheidig-Benatar, Christine; Hernandez-Rivas, Rosaura; Scherf, Artur

2012-01-01

272

'A' forms of RNAs in single strands, duplexes and RNA-DNA hybrids.  

PubMed Central

Helical parameters have been calculated for the 'A' form minimum energy conformations of ApA, CpC, GpG, UpU, GpC and UpA. The helix geometries are base sequence dependent. The single strands are narrower and more tightly wound than that duplex RNA-11 form. 9-12 kcal./mole are needed to convert these single strands to the RNA-11 conformation. However, in some sequences other 'A' type conformers capable of complementary base pairing may be formed at lower energetic cost. There is substantially more base stacking in the calculated single strands than in the RNA-11 conformation. Calculated intrastrand base stacking energies reflect these differences, and also are sequence dependent. The 'A' form RNA subunits differ from the analogous DNAs in possessing a larger rise per residue, needed to accomodate the 2'-OH. RNA-DNA hybrids are consequently more likely to be in the 'A-RNA than in the 'A'-DNA conformation, although the base sequence determines the extent of the preference.

Broyde, S; Hingerty, B

1978-01-01

273

Compositional changes in RNA, DNA and proteins for bacterial adaptation to higher and lower temperatures.  

PubMed

It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed. PMID:12761299

Nakashima, Hiroshi; Fukuchi, Satoshi; Nishikawa, Ken

2003-04-01

274

Adenovirus type 2 early nuclear and mRNA: kinetic estimation of l anf r DNA strand fractions complementary to different abundance classes of viral RNA.  

PubMed

RNA from unfractionated cells, nuclei, and polyribosomes was extracted from adenovirus 2 (Ad2)-infected KB cells early in infection and annealed in vast excess in liquid to purified Ad2 l (heavy) and r (light) [(32)P]DNA strands (specific activity, 3 x 10(6) to 1.5 x 10(7) cpm/mug). The number of abundance classes of Ad2 RNA, their relative concentrations, and the strand fraction from which they arose were determined by a computer-assisted nonlinear regression analysis of hybridization kinetic data. Whole-cell RNA and nuclear RNA annealed to 60 and 40%, respectively, of l and r strands. Well-defined abundance (kinetic) classes were identified: abundant and scarce classes were complementary to 15 to 17 and 40 to 45%, respectively, of l strand, and to 11 to 16 and 17 to 23%, respectively, of r strand. In whole-cell RNA and nuclear RNA the abundant classes were 57 to 208 and 13 to 27 times more concentrated, respectively, than scarce classes. RNA-RNA hybrids were isolated that annealed to about 70% of both strands, indicating that whole-cell RNA and nuclear RNA hybridization values were minimal. Polyribosomal RNA appeared to anneal as three abundance classes to each DNA strand; abundant, scarce, and very scarce classes, respectively, hybridized to 6, 5, and about 10% of l strand and 7 (6 to 8), 10 (8 to 13), and about 19% of r strand. The abundant classes were 41 (11 to 67) times more concentrated than the scarce classes and 10(3) times more concentrated than the very scarce classes. Although the biological significance of these classes is not known, the very scarce classes probably represent nuclear RNA contaminants of polyribosomal RNA. The abundant and scarce classes may comprise mRNA, because together they are complementary to about the same fraction of each DNA strand (11% [10 to 12%] and 17% [14 to 20%] of l and r strands) known to be expressed as early mRNA. Thus, nuclear RNA contains Ad2 RNA sequences not found on polyribosomes; most or all of both DNA strands are transcribed, but only certain transcripts are processed into mRNA. It is not known whether "non-mRNA" transcripts are intermediates in the pathway of early mRNA production. PMID:894792

Wold, W S; Green, M; Brackmann, K H; Devine, C; Cartas, M A

1977-09-01

275

RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element.  

PubMed

R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested. The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat-retrotransposable elements. PMID:7540721

Luan, D D; Eickbush, T H

1995-07-01

276

RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element.  

PubMed Central

R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested. The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat-retrotransposable elements.

Luan, D D; Eickbush, T H

1995-01-01

277

A robust and efficient method for the isolation of DNA-free, pure and intact RNA from Mycobacterium tuberculosis.  

PubMed

We describe a robust method for the isolation of pure, intact RNA suitable for transcriptome studies from mycobacteria with consistent yields of 1 ?g to 3 ?g total RNA per 10(7) cells. The method reduces the use of hazardous chemicals and incorporates protocols for efficient removal of gDNA and rRNA. PMID:23566823

Venkataraman, Balaji; Gupta, Nidhi; Gupta, Amita

2013-04-06

278

Urea Facilitates the Translocation of Single-Stranded DNA and RNA Through the ?-Hemolysin Nanopore  

PubMed Central

Abstract The staphylococcal ?-hemolysin (?HL) protein nanopore is under investigation as a fast, cheap detector for nucleic acid analysis and sequencing. Although discrimination of all four bases of DNA by the ?HL pore has been demonstrated, analysis of single-stranded DNAs and RNAs containing secondary structure mediated by basepairing is prevented because these nucleic acids cannot be translocated through the pore. Here, we show that a structured 95-nucleotide single-stranded DNA and its RNA equivalent are translocated through the ?HL pore in the presence of 4 M urea, a concentration that denatures the secondary structure of the polynucleotides. The ?HL pore is functional even in 7 M urea, and therefore it is easily stable enough for analyses of challenging DNA and RNA species.

Japrung, Deanpen; Henricus, Marsiyana; Li, Qiuhong; Maglia, Giovanni; Bayley, Hagan

2010-01-01

279

Synthesis of digoxigenin-labeled cDNA as a test for mRNA integrity.  

PubMed

The nucleotide analogue digoxigenin-11-dUTP is widely used as a nonradioactive marker in a broad range of techniques including dot blots, Southern and Northern blots, colony and plaque screenings and in situ hybridizations. In this report, we describe the incorporation of this molecule into a cDNA synthesized by reverse transcriptase as a novel application for digoxigenin. This reaction can be performed as a useful control to check the integrity of a bulk mRNA preparation in the construction of a cDNA library, since the ability of mRNA to direct the synthesis of long molecules of first-strand cDNA is a sign of its integrity. PMID:1282346

Soldo, L; Mangano, G; Milanese, C

1992-12-01

280

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.  

PubMed Central

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results. Images

Gohlke, C; Murchie, A I; Lilley, D M; Clegg, R M

1994-01-01

281

Transcription of DNA containing the 5-guanidino-4-nitroimidazole lesion by human RNA polymerase II and bacteriophage T7 RNA polymerase  

Microsoft Academic Search

Damage in transcribed DNA presents a challenge to the cell because it can partially or completely block the progression of an RNA polymerase, interfering with transcription and compromising gene expression. While blockage of RNA polymerase progression is thought to trigger the recruitment of transcription-coupled DNA repair (TCR), bypass of the lesion can also occur, either error-prone or error-free. Error-prone transcription

Alexandra Dimitri; Lei Jia; Vladimir Shafirovich; Nicholas E. Geacintov; Suse Broyde; David A. Scicchitano

2008-01-01

282

External Field Influence on Semiflexible Macromolecules:. Geometric Coupling  

NASA Astrophysics Data System (ADS)

We suggested a geometric approach to address the external field influence on the DNA molecules, described by the WLC model via geometric coupling. It consists in the introduction of the effective metrics depending on the potential of the external field, with further re-definition of the arc-length parameter and of the extrinsic curvatures of the DNA molecules. It yields the nontrivial impact of the external field in the internal energy of macromolecules. We give the Hamiltonian formulation of this model and perform its preliminary analysis in the redefinition of the initial energy density.

Bellucci, Stefano; Mamasakhlisov, Yevgeny; Nersessian, Armen

283

Plant gene therapy: crop varietal improvement through the use of chimaeric RNA\\/DNA oligonucleotide-directed gene targeting  

Microsoft Academic Search

Chimaeric RNA\\/DNA oligonucleotides are synthetic molecules comprised of DNA and RNA bases that assume a self-complementary double hairpin conformation. These molecules have been shown to direct site-specific base changes in plant and animal cells, while not introducing foreign DNA into the genome. Progeny from modified plants derived through the use of this technology have been shown to inherit gene conversions

Gregory D. May; Eric B. Kmiec

2000-01-01

284

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA  

PubMed Central

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5?-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3? side, which contains the Shine–Dalgarno sequence. Here, we show by in vitro gp43–RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine–Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator’s hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5?-AC-3? in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.

Pavlov, Andrey R.; Karam, Jim D.

2000-01-01

285

Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model  

PubMed Central

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

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

2012-01-01

286

Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor.  

PubMed

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is in the form of rate, microV/s, and is directly proportional to the quantity of hybridized DNA captured on the membrane. Use of the larger probes (for beta-actin cDNA target) results in detection of intact plasmid at the level of approximately 106 target molecules; complementary DNA could be detected at similar levels. When the smaller probes are utilized (pGEM or pBSActB targets), plasmid fragmented targets could be detected only at levels 200 times greater than those observed when using the larger RNA probes. PMID:9470098

Dill, K; Chan, S D; Gibbs, T W

1997-12-01

287

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

PubMed

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

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

2012-02-23

288

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.

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

2005-01-01

289

Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine.  

PubMed

5-Hydroxyuridine (5-OHU) is a major lesion of uridine and cytosine produced in RNA by various chemical oxidants. To elucidate its biochemical and biophysical effects on RNA replication, the site-specifically modified oligoribonucleotides containing 5-OHU were synthesized with C5-hydroxy-5'-O-DMTr-2'-TBDMS-uridine phosphoramidite using automated solid phase synthesis. The base-pairing properties of nucleotides opposite 5-OHU in 24 mer oligoribonulcleotides with dNTP were studied using three reverse transcriptases (Super-Script(TM)II-, AMV-, MMLV-RT) in cDNA synthesis. Adenine as well as guanine was incorporated preferentially by all reverse transcriptases. In the UV-melting temperature experiment, the results from the relative stabilities of the base pairs were A : 5-OHU > G : 5-OHU > T : 5-OHU approximately C : 5-OHU. Circular Dichroism (CD) studies showed that DNA-RNA containing 5-OHU heteroduplexes exhibit a similar conformation between the A-type RNA and B-type DNA. These results suggest that 5-OHU from oxidative damage was mainly influenced by adenine mismatch. PMID:19558797

Cui, Song; Kim, Yong-Hoon; Jin, Cheng-Hao; Kim, Sang Kook; Rhee, Man-Hee; Kwon, Oh-Shin; Moon, Byung Jo

2009-06-30

290

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

PubMed Central

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 in which we subjected three ssDNA (?X174, G4, F1) and three ssRNA phages (Q?, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ?X174 and 42 clones of Q? carrying random single-nucleotide substitutions and assayed them for fitness. In ?X174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Q?, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses.

Domingo-Calap, Pilar; Cuevas, Jose M.; Sanjuan, Rafael

2009-01-01

291

Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART.  

PubMed

The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 +/- 2.76) than in DNA (2.88 +/- 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had >or=1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. PMID:18712823

Saracino, Annalisa; Gianotti, Nicola; Marangi, Marianna; Cibelli, Donatella C; Galli, Andrea; Punzi, Grazia; Monno, Laura; Lazzarin, Adriano; Angarano, Gioacchino

2008-10-01

292

DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry  

Microsoft Academic Search

Sets of RNA ladders can be synthesized by transcrip- tion of a bacteriophage-encoded RNA polymerase using 3'-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption\\/ionization time-of-flight mass spec- trometry (MALDI-TOF MS) of chain-terminated RNA

Young-Soo Kwon; Kai Tang; Charles R. Cantor; Hubert Köster; Changwon Kang

2001-01-01

293

In vitro detection of specific mRNA protection or degradation factors using a fluorescence DNA sequencer  

Microsoft Academic Search

A method to detect specific mRNA degradation factors using non-radioactive RNA synthesis has been developed which involves synthesis of RNA by in vitro transcription in the presence of Texas-Red labeled nucleotides. Degradation of RNA synthesized from the chicken aA-globin 3'-UTR region was detected using an automatic fluorescence DNA sequencer. This fluorescence detection method has higher resolution than the conventional slab

Yukinori Eguchi; Tomoko Eguchi

2002-01-01

294

Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.  

PubMed Central

We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods. Images

Gilmour, D S; Lis, J T

1984-01-01

295

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?  

PubMed

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants. PMID:15247494

Vuosku, Jaana; Jaakola, Laura; Jokipii, Soile; Karppinen, Katja; Kämäräinen, Terttu; Pelkonen, Veli-Pekka; Jokela, Anne; Sarjala, Tytti; Hohtola, Anja; Häggman, Hely

2004-07-01

296

Efficient Detection of Three-Dimensional Structural Motifs in Biological Macromolecules by Computer Vision Techniques  

Microsoft Academic Search

Macromolecules carrying biological information often consist of independent modules containing recurring structural motifs. Detection of a specific structural motif within a protein (or DNA) aids in elucidating the role played by the protein (DNA element) and the mechanism of its operation. The number of crystallographically known structures at high resolution is increasing very rapidly. Yet, comparison of three-dimensional structures is

Ruth Nussinov; Haim J. Wolfson

1991-01-01

297

An RNA virus hijacks an incognito function of a DNA repair enzyme.  

PubMed

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold. PMID:22908287

Virgen-Slane, Richard; Rozovics, Janet M; Fitzgerald, Kerry D; Ngo, Tuan; Chou, Wayne; van der Heden van Noort, Gerbrand J; Filippov, Dmitri V; Gershon, Paul D; Semler, Bert L

2012-08-20

298

xUBF, an RNA polymerase I transcription factor, binds crossover DNA with low sequence specificity.  

PubMed Central

Xenopus UBF (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. These include a short basic region adjacent to a dimer motif plus five high-mobility-group (HMG) boxes. All of these DNA-binding motifs exhibit low sequence specificity, whether assayed singly or together. In contrast, the HMG boxes recognize DNA structure that is formed when two double helices are crossed over each other. HMG box 1, in particular, requires association of two double helices before it will bind and, either by itself or in the context of the intact protein, will loop DNA and organize it into higher-order structures. We discuss how this mode of binding affects the function of xUBF as a transcription factor. Images

Hu, C H; McStay, B; Jeong, S W; Reeder, R H

1994-01-01

299

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.  

PubMed Central

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers. Images

Vishwanatha, J K; Yamaguchi, M; DePamphilis, M L; Baril, E F

1986-01-01

300

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

301

The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates  

Microsoft Academic Search

Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochon- drial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p.

Jean-Baptiste Boule ´; Virginia A. Zakian

2007-01-01

302

Hypoxia-induced growth limitation of juvenile fishes in an estuarine nursery: assessment of small-scale temporal dynamics using RNA:DNA  

Microsoft Academic Search

The ratio of RNA to DNA (RNA:DNA) in white muscle tissue of juvenile summer flounder (Paralichthys den- tatus) and weakfish (Cynoscion regalis) was used as a proxy for recent growth rate in an estuarine nursery. Variability in RNA:DNA was examined relative to temporal changes in temperature and dissolved oxygen (DO). Initial laboratory ex- periments indicated (i) a strong positive relationship

Kevin L. Stierhoff; Timothy E. Targett; James H. Power

2009-01-01

303

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

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

1986-01-01

304

p53-Dependent p21 mRNA Elongation Is Impaired when DNA Replication Is Stalled? †  

PubMed Central

We have previously reported that when DNA replication is blocked in some human cell lines, p53 is impaired in its ability to induce a subset of its key target genes, including p21WAF1/CIP1. Here, we investigated the reason for this impairment by comparing the effects of two agents, hydroxyurea (HU), which arrests cells in early S phase and impairs induction of p21, and daunorubicin, which causes a G2 block and leads to robust activation of p21 by p53. HU treatment was shown to inhibit p21 mRNA transcription rather than alter its mRNA stability. Nevertheless, chromatin immunoprecipitation assays revealed that HU impacts neither p53 binding nor acetylation of histones H3 and H4 within the p21 promoter. Furthermore, recruitment of the TFIID/TATA-binding protein complex and the large subunit of RNA polymerase II (RNA Pol II) are equivalent after HU and daunorubicin treatments. Relative to daunorubicin treatment, however, transcription elongation of the p21 gene is significantly impaired in cells treated with HU, as evidenced by reduced occupancy of RNA Pol II at regions downstream of the start site. Likewise, in the p21 downstream region after administration of HU, there is less of a specifically phosphorylated form of RNA Pol II (Pol II-C-terminal domain serine 2P) which occurs only when the polymerase is elongating RNA. We propose that while the DNA replication checkpoint is unlikely to regulate the assembly of a p21 promoter initiation complex, it signals to one or more factors involved in the process of transcriptional elongation.

Mattia, Melissa; Gottifredi, Vanesa; McKinney, Kristine; Prives, Carol

2007-01-01

305

Ultrasensitive DNA Microarray Biosensing via in situ RNA Transcription-Based Amplification and Nanoparticle-Enhanced SPR Imaging  

PubMed Central

DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto a ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this “generator” element is then detected by specific adsorption onto a second adjacent “detector” element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of complementary DNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 ?L (25 zeptomoles).

Sendroiu, Iuliana E.; Gifford, Lida K.; Luptak, Andrej; Corn, Robert M.

2011-01-01

306

Cloning and physical mapping of DNA complementary to potato leafroll virus RNA  

SciTech Connect

Potato leafroll virus (PLRV) was aphid-transmitted from potato (Solanum tuberosum cultivar Russett Burbank) to ground cherry (Physalis floridana), where it was maintained by serial aphid transmission. Serological and plant differential tests indicated that the isolate was not contaminated with beet western yellows virus. Purified PLRV RNA was poly(A)-tailed in vitro and used as a template for reverse transcriptase, primed with oligo(dT). Alkaline gel electrophoresis of /sup 32/P-labeled first-strand complementary DNA (cDNA) indicated a major size range of 0.1 to 3.5 kilobases (kb). A small percentage of transcripts corresponded to full length PLRV RNA. Following RNase H and DNA polymerase I-mediated second strand synthesis, double-stranded cDNA was cloned into the Pst I site of the plasmid pUC9 using oligo (dC)-oligo(dG) tailing methodology. Escherichia coli JM109 transformants were screened with first-strand /sup 32/P-cDNA in colony hybridization experiments to confirm that recombinants contained PLRV-specific sequences.

Smith, O.P.

1987-01-01

307

RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves  

PubMed Central

Cauliflower mosaic virus (CaMV) is a plant DNA with an 8-kb circular double-stranded genome. CaMV-specific DNA and RNA molecules present in infected Brassica cells share some structural features with DNAs and RNAs of retroviruses and hepatitis B virus. This led to the hypothesis that CaMV replication occurs via reverse transcription of an RNA intermediate. Here we report the first characterization of a new DNA polymerase activity, specific to CaMV-infected tissues. A subcellular fraction of infected cells shows capacity to copy poly(C) and the heteropolymeric regions of natural mRNAs. Chromatographic isolation of the poly(C)-dependent activity clearly establishes that it is distinct from the classical ?-like DNA polymerases previously described in plant cells. The significant homology observed between defined regions of the Moloney murine leukemia virus (MMLV) polymerase and CaMV unassigned gene V product favours the idea that the reverse transcriptase-like DNA polymerase detected in infected cells is a virus-encoded enzyme.

Volovitch, M.; Modjtahedi, N.; Yot, P.; Brun, G.

1984-01-01

308

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

PubMed

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

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

2008-10-07

309

Isolation and characterization of homologous TRBP cDNA for RNA interference in Penaeus monodon.  

PubMed

The transactivation response RNA-binding protein (TRBP) interacts with Dicer and binds to double-stranded RNA as a critical component of the RNA-induced silencing complex, which is a key complex in the RNA interference pathway. The full-length cDNA of TRBP from the tiger prawn, Penaeus monodon, (PmTRBP; 1548 bp long with a 1029 bp coding region) was isolated. The encoded polypeptide of 343 amino acids had a predicted molecular mass of 36.8 kDa. Sequence homology and phylogenetic analysis indicated that PmTRBP was evolutionarily closest to TRBP1 from Litopenaeus vannamei, with the three double-stranded RNA-binding motifs that were typical of the TRBP family. Tissue expression profile analysis by quantitative real-time reverse transcription polymerase chain reaction showed that PmTRBP1 was constitutively expressed in all the examined tissues, with a predominant expression in the lymphatic organs and with the weakest expression in the ovaries. Significantly upregulated PmTRBP1 expression was elicited by systemic injections of Staphylococcus aureus, Vibrio vulnificus, and white spot syndrome virus, thereby revealing its pathogen inducibility. Furthermore, exogenous viral nucleoside analogs (high-molecular-weight poly(I:C) dsRNAs as well as R484 single-stranded RNA) were remarkably induced PmTRBP1 transcription at 48 h and 9 h post-injection, respectively, which suggested that PmTRBP1 might function in tiger prawn antibacterial and antiviral response. PMID:23207479

Yang, Lishi; Li, Xiaolan; Huang, Jianhua; Zhou, Falin; Su, Tianfeng; Jiang, Shigui

2012-11-30

310

Transcriptional enhancement of the Listeria monocytogenes PCR and simple immunoenzymatic assay of the product using anti-RNA:DNA antibodies.  

PubMed Central

A method was developed to enhance the sensitivity of a Listeria monocytogenes PCR detection system by in vitro transcription of amplicons incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. The resulting transcript can be detected by hybridization with a DNA probe immobilized in the wells of a microtiter plate, followed by immunoenzymatic assay of the RNA-DNA hybrids with an anti-RNA-DNA hybrid antibody. This highly sensitive method was reactive in the assay of various L. monocytogenes isolates but not with other Listeria or non-Listeria species. Images

Blais, B W

1994-01-01

311

DNA and RNA analyses in detection of genetic predisposition to cancer  

PubMed Central

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

2012-01-01

312

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

PubMed

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

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

2013-02-14

313

Molecular cloning and sequencing of DNA complementary to chicken liver fatty acid synthase mRNA.  

PubMed Central

The cDNA corresponding to 4.18 kilobases (kb) of the mRNA of chicken liver fatty acid synthase has been cloned and sequenced. The cDNA corresponds to the 3' end of the mRNA and consists of a 1.87-kb noncoding tail and a 2.31-kb region encoding 769 amino acids of the C terminus of the enzyme. The thioesterase at the C terminus, preceded by the acyl carrier protein, can be identified from known amino acid sequences. However, the identity of the enzymes N terminal to the acyl carrier protein could not be ascertained. The partial amino acid sequence of the chicken liver fatty acid synthase shows greater than 70% similarity with the rat mammary gland enzyme.

Yuan, Z Y; Liu, W; Hammes, G G

1988-01-01

314

Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.  

PubMed

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity. PMID:19633128

Peleg, Ofer; Baneth, Gad; Eyal, Osnat; Inbar, Jacob; Harrus, Shimon

2009-07-24

315

Use of Chimeric DNA-RNA Primers in Quantitative PCR for Detection of Ehrlichia canis and Babesia canis? †  

PubMed Central

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine ?-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.

Peleg, Ofer; Baneth, Gad; Eyal, Osnat; Inbar, Jacob; Harrus, Shimon

2009-01-01

316

Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases  

SciTech Connect

To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

1985-07-01

317

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

PubMed

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

Chícharo, Maria A; Chícharo, Luis

2008-08-20

318

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

319

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

320

Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid  

PubMed Central

Background The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation. Conclusion This work contributes to the development of new alternatives for controlling the fungal agent of witches' broom disease.

Andrade, Bruno S; Taranto, Alex G; Goes-Neto, Aristoteles; Duarte, Angelo A

2009-01-01

321

Cross section calculations for electron scattering from DNA and RNA bases  

Microsoft Academic Search

Differential and integral cross sections for elastic electron collisions with uracil, cytosine, guanine, adenine and thymine have been calculated using the independent atom method with a static-polarization model potential for incident energies ranging from 50 to 4000 eV. Total cross sections for single electron-impact ionization of selected DNA and RNA bases have also been calculated with the binary-encounter-Bethe model from the

Pawe? Mo?ejko; Léon Sanche

2003-01-01

322

Chemically Programmed Polymers for Targeted DNA and siRNA Transfection  

Microsoft Academic Search

\\u000a Plasmid DNA and siRNA have a large potential for use as therapeutic nucleic acids in medicine. The way to the target cell\\u000a and its proper compartment is full of obstacles. Polymeric carriers help to overcome the encountered barriers. Cationic polymers\\u000a can interact with the nucleic acid in a nondamaging way but still require optimization with regard to transfer efficiency\\u000a and

Eveline Edith Salcher; Ernst Wagner

323

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.

Chicharo, Maria Alexandra; Chicharo, Luis

2008-01-01

324

On the Prebiotic Synthesis of Nucleobases, Nucleotides, Oligonucleotides, Pre-RNA and Pre-DNA Molecules  

Microsoft Academic Search

All the strategies for the prebiotic syntheses of RNA and DNA assume the adequate availability\\u000a of the presumptive precursors such as purine and pyrimidine nucleic bases, nucleosides and nucleotides.\\u000a Polymerization of activated nucleotides probably furnished the first informational oligonucleotides.\\u000a The formation of these precursors from mixtures of simple gases was shown to occur in a variety\\u000a of conditions including UV-irradiation, electric

Raffaele Saladino; Claudia Crestini; Giovanna Costanzo; Ernesto DiMauro

325

Type C RNA Virus Proteins: Lack of Binding Specificity to Host Cell Chromosomal DNA in vitro  

Microsoft Academic Search

Through the use of two different DNA-protein reconstitution methods, we examined the potential role of type C RNA tumor virus proteins as putative regulatory agents in the control of virus expression. Rauscher murine leukemia virus, purified from chronically infected rat cell cultures, was iodinated in vitro with [125I], and dissociated in a nondetergent high-ionic-strength urea-containing buffer. The chemically separated [125I]-labeled

Kathryn Miles; Stephen A. Schwartz

1979-01-01

326

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm  

Microsoft Academic Search

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a 5 ZAP II library prepared from poly (A+) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm

T. Abe; H. Niiyama; T. Sasahara

2002-01-01

327

Restriction enzyme mapping the ribosomal RNA genes in Solanum tuberosum: Potato rDNA restriction enzyme map  

Microsoft Academic Search

A restriction enzyme map of the ribosomal RNA genes (rDNA) in Solanum tuberosum cultivars Golden Wonder and Desiree has been constructed. An heterologous probe pTa 71 containing the rDNA derived from wheat was used to detect and map the corresponding region in potato genomic DNA fragments. rDNA repeats of cultivars Desiree and Golden Wonder are similar with respect to their

K. Harding

1991-01-01

328

DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes  

Microsoft Academic Search

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level\\u000a identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial\\u000a COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish\\u000a these

Yanwei Feng; Qi Li; Lingfeng Kong; Xiaodong Zheng

2011-01-01

329

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

PubMed Central

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

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

2013-01-01

330

[RNA, unlike DNA, binds to aurintricarboxylic acid via covalent bonds: molecular-biological and spectroscopic evidence].  

PubMed

A selective interaction of ATA with RNA, unlike DNA, when isolating nucleic acids from plant materials was established. The data concerning the binding strength of highly purified RNAATA and DNAATA preparations to nitrocellulose and nylon filters under conditions of high ionic strength are presented. The interrelation of ATA RNA-tropism and absence of adsorption capability of chemical modified RNAATA the backbone was observed. Using IR spectroscopy under the procedure of multi-broken complete light reflection, a formation of ATA and RNA complex was fixed via phosphoric-ether bond (P-O-C), which was absent in the case of DNAATA. The obtained data raise the problem of RNAATA application in molecular biology experiments. PMID:12924021

Martynenko, E I; Negrebetskaia, E N; Stepaniugin, A V; Samo?lenko, S A; Govorun, D N

331

Delivery of Drugs and Macromolecules to Mitochondria  

PubMed Central

Mitochondria is where the bulk of the cell’s ATP is produced. Mutations occur to genes coding for members of the complexes involved in energy production. Some are a result of damages to nuclear coded genes and others to mitochondrial coded genes. This review describes approaches to bring small molecules, proteins and RNA/DNA into mitochondria. The purpose is to repair damaged genes as well as to interrupt mitochondrial function including energy production, oxygen radical formation and the apoptotic pathway.

Mukhopadhyay, Abhijit; Weiner, Henry

2008-01-01

332

Effect of multiple freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology.  

PubMed

Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (P < 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable. PMID:10325307

Krajden, M; Minor, J M; Rifkin, O; Comanor, L

1999-06-01

333

Adsorption of DNA/RNA nucleobases on hexagonal boron nitride sheet: an ab initio study.  

PubMed

Our ab initio calculations indicate that the interaction of deoxyribonucleic/ribonucleic acid (DNA/RNA) nucleobases [guanine (G), adenine (A), thymine (T), cytosine (C), and uracil (U)] with the hexagonal boron nitride (h-BN) sheet, a polar but chemically inert surface, is governed by mutual polarization. Unlike the case of graphene, all nucleobases exhibit the same stacking arrangement on the h-BN sheet due to polarization effects: the anions (N and O atoms) of nucleobases prefer to stay on top of cations (B) of the substrate as far as possible, regardless of the biological properties of nucleobases. The adsorption energies, ranging from 0.5 eV to 0.69 eV, increase in the order of U, C, T, A and G, which can be attributed to different side groups or atoms of nucleobases. The fundamental nature of DNA/RNA nucleobases and h-BN sheet remains unchanged upon adsorption, suggesting that the h-BN sheet is a promising template for DNA/RNA-related research, such as self-assembly. PMID:21637870

Lin, Qing; Zou, Xiaolong; Zhou, Gang; Liu, Rui; Wu, Jian; Li, Jia; Duan, Wenhui

2011-06-03

334

RNA and DNA synthesis of epidermal basal cells after wounding. Comparison of vital and postmortem investigations.  

PubMed

Incision wounds were made on both of the pinnae of each rat, and two biopsies from both the ears were taken for examination after different survival times of the wounds. Two biopsies were taken from each ear, four from each animal, two intravitally and two postmortem after 24 hours storage at 8 degrees C. One each of the intravital and one each of the postmortem biopsies were prepared and evaluated for quantification of RNA and DNA synthesis rate using an in vitro incorporation model with 3H-cytidine and bromodeoxyuridine (BrdU) as markers. The intravital specimens showed a significant increase in 3H-cytidine incorporation in the basal cell layer after survival times of 10 to 24 hours. No increase in the rate of RNA synthesis in the basal cell layer as a function of wound age was seen in postmortem specimens. In both intravital and postmortem biopsies the labelling indices after BrdU exposition increased significantly in the period from 32 to 60 hours post-injury. This suggest that DNA synthesis induced during life continues after death. Applied to forensic practice, these findings point to the possibility of determining the vitality of a wound in postmortem tissue. The RNA synthesis, obviously, precedes the DNA synthesis after mechanical trauma. PMID:9314058

Oehmichen, M; Lagodka, T; Cröpelin, A

1997-08-01

335

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning  

SciTech Connect

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

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

1990-10-01

336

A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.  

PubMed

Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. PMID:20394837

Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

2010-04-13

337

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.

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

2010-01-01

338

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

PubMed Central

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

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

2013-01-01

339

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

PubMed

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

Yeom, Ji-Hyun; Ryou, Sang-Mi; Won, Miae; Park, Mira; Bae, Jeehyeon; Lee, Kangseok

2013-09-20

340

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere  

PubMed Central

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Moran-Jones, Kim; Wayman, Lyndal; Kennedy, Derek D.; Reddel, Roger R.; Sara, Sergio; Snee, Mark J.; Smith, Ross

2005-01-01

341

Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases  

Microsoft Academic Search

BACKGROUND: The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. RESULTS: Using extensive

Lakshminarayan M Iyer; Eugene V Koonin; L Aravind

2003-01-01

342

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.

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

2012-01-01

343

Influence of the ?-? interaction on the hydrogen bonding capacity of stacked DNA/RNA bases  

PubMed Central

The interplay between aromatic stacking and hydrogen bonding in nucleobases has been investigated via high-level quantum chemical calculations. The experimentally observed stacking arrangement between consecutive bases in DNA and RNA/DNA double helices is shown to enhance their hydrogen bonding ability as opposed to gas phase optimized complexes. This phenomenon results from more repulsive electrostatic interactions as is demonstrated in a model system of cytosine stacked offset-parallel with substituted benzenes. Therefore, the H-bonding capacity of the N3 and O2 atoms of cytosine increases linearly with the electrostatic repulsion between the stacked rings. The local hardness, a density functional theory-based reactivity descriptor, appears to be a key index associated with the molecular electrostatic potential (MEP) minima around H-bond accepting atoms, and is inversely proportional to the electrostatic interaction between stacked molecules. Finally, the MEP minima on surfaces around the bases in experimental structures of DNA and RNA–DNA double helices show that their hydrogen bonding capacity increases when taking more neighboring (intra-strand) stacking partners into account.

Mignon, Pierre; Loverix, Stefan; Steyaert, Jan; Geerlings, Paul

2005-01-01

344

Trial by fire: are the crystals macromolecules?  

PubMed Central

Protein crystallization screens frequently yield salt crystals as well as protein crystals. A simple method for determining whether a crystal is composed of salt or macromolecules is suggested. A drop containing one or more crystals is transferred to a glass cover slip and the cover slip is then passed through the flame of a Bunsen burner. Macromolecule crystals are destroyed by this treatment, while salt crystals generally remain. The test can be performed after other commonly used tests such as crushing and staining.

Raghunathan, Kannan; Harris, Paul T.; Arvidson, Dennis N.

2010-01-01

345

Molecular weights of lignite macromolecules  

SciTech Connect

A sample of the base-extractable product from the 4-nitroperbenzoic acid oxidation of lignite was converted to the methylated derivative with dimethyl sulfate followed by diazomethane in DMF-ether, and was reductively acetylated with zinc dust in acetic anhydride as in the humic acid derivatization above. A light tan THF-soluble material was obtained. The molecular weight was determined in the static LALLS cell in THF solution. A linear relationship was obtained in the Kc/R/sub theta/ versus c plot (r/sup 2/ = 0.95). The molecular weight obtained for the derivative of the oxidation product (1.3 x 10/sup 6/) was the same as the value obtained for the humic acid derivative. The reductively acetylated oxidation product was also examined with the SEC-LALLS-RI system. A symmetrical peak was observed for the LALLS response, and a nonsymmetrical peak for the RI response. Somewhat lower values for the number average, weight average and z average molecular weights were calculated from these data (Mn = 6.5 x 10/sup 5/, Mw = 7.8 x 10/sup 5/, Mz = 9.1 x 10/sup 5/). These data indicate that by oxidatively cleaving the benzyl ether groups with the peracid, a substantial macromolecule is produced which is comparable in size to that obtained by simple base extraction of the lignite. This implies that there is some basic unit of this molecular weight making up the coal structure, and that in base extraction some of these units are released, whereas in peracid oxidation a large number of them are released by scission of cross-linking bonds. 6 refs., 4 figs.

Olson, E.S.; Diehl, J.W.; Froehlich, M.L.

1985-10-01

346

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.

347

DNA's Liaison with RNA Polymerase Physical Consequences of a Twisted Relationship  

NASA Astrophysics Data System (ADS)

RNA polymerase is the molecular motor that performs the fundamental process of transcription. Besides being the key- protagonist of gene regulation it is one of the most powerful nano-mechanical force generators known inside the cell. The fact that polymerase strictly tracks only one of DNA's strands together with DNA's helical geometry induces a force-to-torque transmission, with several important biological consequences like the ``twin supercoil domain'' effect and remote torsional interaction of genes. In the first part of the talk we theoretically explore the mechanisms of non-equilibrium transport of twist generated by a moving polymerase. We show that these equations are intrinsically non-linear in the crowded cellular environment and lead to peculiar effects like self-confinement of torsional strain by generation of alternative DNA structures like cruciforms. We demonstrate how the asymmetric conformational properties of DNA lead to a ``torsional diode'' effect, i.e. a rectification of polymerase-generated twist currents of different signs. In the second part we explore the possibility of exploiting the polymerase as a powerful workhorse for nanomechanical devices. We propose simple and easy to assemble arrangements of DNA templates interconnected by strand-hybridization that when transcribed by the polymerase linearly contract by tenfold. We show that the typical forces generated by such ``DNA stress fibers'' are in the piconewton range. We discuss their kinetics of contraction and relaxation and draw parallels to natural muscle fiber design.

Kulic, Igor; Nelson, Phil

2006-03-01

348

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

349

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.

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

2012-01-01

350

Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents  

SciTech Connect

A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells). Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium. The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours. No significant DNA amplification was detectable in the treated cells.

Laval, F. (U247 INSERM, Institut Gustave Roussy, Villejuif (France))

1991-05-15

351

Selective Nucleic Acid Removal via Exclusion (SNARE): Capturing mRNA and DNA from a Single Sample.  

PubMed

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

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

2013-09-26

352

Characterization of potentially functional 5S rRNA-encoding genes within ribosomal DNA repeats of the nematode Meloidogyne arenaria.  

PubMed

In the plant-parasitic nematode Meloidogyne arenaria, isolated rDNA repeats show length heterogeneity, and are unusual in that they contain putative 5S ribosomal RNA pseudogenes [Vahidi et al., J. Mol. Evol. 27 (1988) 222-227]. Potentially functional 5S rRNA-encoding genes can also be identified in various rDNA repeats, which appear to be tandemly organized in the genome. PMID:1748312

Vahidi, H; Purac, A; LeBlanc, J M; Honda, B M

1991-12-15

353

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

354

Synthesis, DNA\\/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene  

Microsoft Academic Search

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5–10 and 15) has been synthesized. Compounds 5–10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA, whereby most of them form stacked dimers along the RNA double helix. The only exception is compound 15 which, due

Ivana Stoli?; Katarina Miškovi?; Ivo Piantanida; Mirela Baus Lon?ar; Ljubica Glavaš-Obrovac; Miroslav Baji?

2011-01-01

355

Mutational Analysis of the mRNA Operator for T4 DNA Polymerase  

PubMed Central

Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C->A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A->U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

Andrake, M. D.; Karam, J. D.

1991-01-01

356

A Three-Helix Junction Is the Interface between Two Functional Domains of Prohead RNA in ?29 DNA Packaging  

PubMed Central

The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage ?29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.

Zhao, Wei; Saha, Mitul; Ke, Ailong; Morais, Marc C.; Jardine, Paul J.

2012-01-01

357

Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope.  

PubMed

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/MgCl2 are shown before and after Escherichia coli RNA polymerase holoenzyme is injected in the SFM liquid chamber. The protein can recognize and bind to these DNA fragments within several seconds after injection, suggesting that the protein and the DNA retain their native configuration after deposition and during SFM imaging. A time-lapse sequence depicting the process of assembly of RNA polymerase-DNA complexes is shown. These results represent the first step for acquiring the capabilities to monitor complex biomolecular processes as they take place in ionic solutions and to characterize their spatial organization. PMID:7809148

Guthold, M; Bezanilla, M; Erie, D A; Jenkins, B; Hansma, H G; Bustamante, C

1994-12-20

358

Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells  

PubMed Central

Virus infection triggers IFN immune defenses in infected cells in part through viral nucleic acid interactions, but the pathways by which dsDNA and DNA viruses trigger innate defenses are only partially understood. Here we present evidence that both retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS) are required for dsDNA-induced IFN-? promoter activation in a human hepatoma cell line (Huh-7), and that activation is efficiently blocked by the hepatitis C virus NS3/4A protease, which is known to block dsRNA signaling by cleaving MAVS. These findings suggest that dsDNA and dsRNA share a common pathway to trigger the innate antiviral defense response in human cells, although dsDNA appears to trigger that pathway upstream of the dsRNA-interacting protein RIG-I.

Cheng, Guofeng; Zhong, Jin; Chung, Josan; Chisari, Francis V.

2007-01-01

359

cDNA Cloning of Goat DNA Methyltransferase 1, Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos  

Microsoft Academic Search

This study was designed to clone cDNA of goat DNA methyltransferase 1 (DNMT1) gene, to screen an effective shRNA-producing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells. In this study, PCR primers were designed against regions of high homology between bovine and sheep

Jie LAN; Yong-li SONG; Song HUA; Yong-gang LIU; Jun LIU; Hai-lin ZHANG; Yong ZHANG

2010-01-01

360

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

PubMed Central

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.

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

2012-01-01

361

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines.  

PubMed

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines. PMID:15550120

Vajdy, Michael; Srivastava, Indresh; Polo, John; Donnelly, John; O'Hagan, Derek; Singh, Manmohan

2004-12-01

362

A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II.  

PubMed

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

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

2013-04-16

363

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

PubMed Central

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

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

2013-01-01

364

DNA polymerase beta mRNA determination by relative quantitative RT-PCR from Leishmania infantum intracellular amastigotes  

Microsoft Academic Search

Gene expression level is extremely difficult to assess in the intracellular form of Leishmania infantum, the amastigote, due to host mRNA contamination, low supply of parasites and stress degradation problems. We obtained and analyzed L. infantum DNA polymerase # (Li Pol #) , a suitable enzyme for differential expression studies. The amount of Li Pol # mRNA was determined in

M. Ramiro; T. Hanke; S. Taladriz; V. Larraga

2002-01-01

365

Relationships between growth rate and RNA, DNA, protein and dry weight in Artemia salina and Euchaeta elongata  

Microsoft Academic Search

The concentrations of RNA, DNA and protein, and the dry weight in 3 cultures of the anostracan Artemia salina (L.) were measured to investigate the usefulness of the RNA-growth relationship in estimating growth or productivity. Similar analyses were performed on Copepodid stages III to VI of the calanoid copepod Euchaeta elongata Esterly collected periodically over 7 months in Haro Strait

M. J. Dagg; J. L. Littlepage

1972-01-01

366

Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses  

Microsoft Academic Search

The genetic study of double-stranded (ds) RNA viruses by sequence analyses of full-length genome segments, or entire viral genomes, has been restricted by the technical difficulties involved in analyses of dsRNA templates. This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies. These methods include an improved version of the ‘Single

Sushila Maan; Shujing Rao; Narender Singh Maan; Simon John Anthony; Houssam Attoui; Alan Richard Samuel; Peter Paul Clement Mertens

2007-01-01

367

Combined Measurement of Serum DNA and Urine VP1 Messenger RNA in Monitoring BK Virus Replication in Kidney Graft Recipients  

Microsoft Academic Search

Evaluation of BK virus replication is a fundamental tool in the monitoring of renal transplant recipients. Herein, we investigated the role of urine VP1 messenger RNA (mRNA) quantification and combined measurement of serum DNA and urine VP1 mRNA in 428 kidney allograft recipients. BK viremia and viruria were detected in 24 (5.6%) and 54 (12.6%) patients, respectively. A diagnosis of

S. Astegiano; M. Bergallo; M. E. Terlizzi; F. Sidoti; S. Gambarino; M. Messina; C. Costa; G. P. Segoloni; R. Cavallo

2011-01-01

368

Role of DNA-Dependent RNA Polymerases II and III in Transcription of the Adenovirus Genome Late in Productive Infection  

Microsoft Academic Search

DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of alpha -amanitin (50% inhibition at 0.02 mu g\\/ml), RNA polymerase III was completely sensitive to high concentrations

Roberto Weinmann; Heschel J. Raskas; Robert G. Roeder

1974-01-01

369

Co-delivery of small interfering RNA and plasmid DNA using a polymeric vector incorporating endosomolytic oligomeric sulfonamide  

PubMed Central

Cationic polymers are potential intracellular carriers for small interfering RNA (siRNA). The short and rigid nature of an siRNA chain often results in larger and more loosely packed particles compared to plasmid DNA (pDNA) after complexing with carrier polycations, and in turn, poor silencing effects are seen against the target mRNAs. A helper polyanion, pDNA, was incorporated along with siRNA to form compact nanosized polyplexes. At C/A (cation/anion) ratios of 2 and 5, poly(L-lysine) (PLL)/siRNA-pGFP and PLL/siRNA-pGFP-OSDZ (oligomeric sulfadiazine (OSDZ) for endosomolysis) complexes produced particles 90–150 nm in size with a 15–45 mV surface charge, while PLL/siRNA complexes yielded particles 1–2 ?m in size at the same C/A ratios. The PLL/siRNA-pGFP (C/A 2) complexes showed significantly higher specific gene silencing (50–90% vs. 10–25%) than the complexes formed at C/A 5. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes improved the specific gene silencing (90%) more dramatically than PLL/siRNA-pGFP (C/A 2) complexes (50%), demonstrating a potential role for OSDZ. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes sustained higher specific gene silencing compared with PLL/siRNA-pGFP (C/A 2) complexes. Other oligomeric sulfonamides (OSA) with varying pKa used in PLL/siRNA-pGFP-OSA complexes also caused effective gene silencing. The pGFP in the PLL/siRNA-pGFP complexes successfully expressed GFP protein without interfering with the siRNA. In conclusion, this study demonstrates that long pDNA helps effectively form nanosized siRNA particles and that OSA enhances specific gene silencing. In a single nucleic acid carrier formulation, co-delivery of siRNA and pDNA is feasible to maximize therapeutic effects or to include therapeutic or diagnostic functionalities.

Kang, Han Chang; Bae, You Han

2011-01-01

370

Route of glucocorticoid-induced macromolecules across the nuclear envelope as viewed by atomic force microscopy  

Microsoft Academic Search

Glucocorticoids are vital steroid hormones. The physiologic activities of these hydrophobic molecules predominantly require translocation of glucocorticoid-initiated macromolecules (GIMs), proteins and mRNA transcripts, in and out of the nucleus, respectively. The bidirectional transport of GIMs is mediated by nuclear pore complexes (NPCs) that span the nuclear envelope at regular distances. The transport proceeds through the NPC central channel, whose interior

Victor Shahin

2006-01-01

371

Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis.  

PubMed Central

We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.

Moran, S; Ren, R X; Sheils, C J; Rumney, S; Kool, E T

1996-01-01

372

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning  

SciTech Connect

The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using (/sup 32/P)-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase.

Han, J.H.; Stratowa, C.; Rutter, W.J.

1987-03-24

373

Determination of aflatoxin B1 biotransformation and binding to hepatic macromolecules in human precision liver slices.  

PubMed

Although epidemiological studies suggest that aflatoxin B1 (AFB1) is a human carcinogen, at least in the presence of hepatitis B virus infection, animal studies have demonstrated large differences in species sensitivity to AFB1, and the sensitivity of humans relative to experimental animals remains unclear. The purpose of this study was to determine the profile of AFB1 metabolism and the extent of AFB1 binding to cell macromolecules in human liver slices under experimental conditions that would allow direct comparison to similar endpoints in the rat, a species sensitive to the carcinogenic actions of AFB1. Liver slices were prepared from three individual human liver samples with a Krumdieck tissue slicer and incubated with 0.5 microM [3H]AFB1 for 2 hr. Significant interindividual variations were observed in the rates of oxidative metabolite formation and in specific binding to cell macromolecules. The rates of oxidative metabolism of AFB1 to AFQ1, AFP1, and AFM1 in the three human liver samples were similar to those previously observed in rat liver slices. AFB1-GSH conjugate formation was not detected in any of the human liver samples, and yet specific binding of AFB1 to cell macromolecules was considerably lower in the human liver slices relative to that in rat liver slices. AFB1-DNA binding levels ranged from 3 to 26% of control rat and AFB1-RNA binding levels ranged from 25 to 49% of control rat. The AFB1-protein binding level in the one human sample measured was 20% of that observed for control rat. While these results suggest that humans do not form as much AFBO as the rat, they are also consistent with the hypothesis that humans do not possess GST isozyme(s) with high specific activity toward AFBO. Significant individual differences in AFB1 metabolism and binding between humans suggest the presence of genetic and/or environmental factors that may confer large variability in susceptibility to AFB1. PMID:8560461

Heinonen, J T; Fisher, R; Brendel, K; Eaton, D L

1996-01-01

374

Arabidopsis C-Terminal Domain Phosphatase-Like 1 Functions in miRNA Accumulation and DNA Methylation  

PubMed Central

Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that cpl1 was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of ros1 suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, cpl1 reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.

Jeong, In Sil; Aksoy, Emre; Fukudome, Akihito; Akhter, Salina; Hiraguri, Akihiro; Fukuhara, Toshiyuki; Bahk, Jeong Dong; Koiwa, Hisashi

2013-01-01

375

Thermodynamics of RNA/DNA hybridization in high-density oligonucleotide microarrays  

NASA Astrophysics Data System (ADS)

We analyze a series of publicly available controlled experiments (Latin square) on Affymetrix high-density oligonucleotide microarrays using a simple physical model of the hybridization process. We plot for each gene the signal intensity vs. the hybridization free energy of RNA/DNA duplexes in solution, for perfect matching and mismatching probes. Both values tend to align on a single master curve in good agreement with Langmuir adsorption theory, provided one takes into account the decrease of the effective target concentration due to target target hybridization in solution. We give an example of a deviation from the expected thermodynamical behavior for the probe set 1091_at due to annotation problems, i.e., the surface-bound probe is not the exact complement of the target RNA sequence, because of errors present in public databases at the time when the array was designed. We show that the parametrization of the experimental data with RNA/DNA free energy improves the quality of the fits and enhances the stability of the fitting parameters compared to previous studies.

Carlon, Enrico; Heim, Thomas

2006-04-01

376

IDN2 and Its Paralogs Form a Complex Required for RNA-Directed DNA Methylation  

PubMed Central

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM.

Zhang, Cui-Jun; Ning, Yong-Qiang; Zhang, Su-Wei; Chen, Qing; Shao, Chang-Rong; Guo, Yan-Wu; Zhou, Jin-Xing; Li, Lin; Chen, She; He, Xin-Jian

2012-01-01

377

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions  

SciTech Connect

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(AACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3{prime}-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3{prime} end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1{prime}-H2{prime} J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2{prime}-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but these results differ somewhat from previous theoretical model studies.

Chou, Shanho (Howard Hughes Medical Inst., Seattle, WA (United States) Univ. of Washington, Seattle (United States)); Flynn, P.; Wang, A.; Reid, B. (Univ. of Washington, Seattle (United States))

1991-05-28

378

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

PubMed

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

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

2013-05-29

379

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.  

PubMed

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 ?l saliva, 5-0.01 ?l semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 ?l saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies. PMID:23165093

Haas, C; Hanson, E; Anjos, M J; Banemann, R; Berti, A; Borges, E; Carracedo, A; Carvalho, M; Courts, C; De Cock, G; Dötsch, M; Flynn, S; Gomes, I; Hollard, C; Hjort, B; Hoff-Olsen, P; Hríbiková, K; Lindenbergh, A; Ludes, B; Marońas, O; McCallum, N; Moore, D; Morling, N; Niederstätter, H; Noel, F; Parson, W; Popielarz, C; Rapone, C; Roeder, A D; Ruiz, Y; Sauer, E; Schneider, P M; Sijen, T; Court, D Syndercombe; Sviežená, B; Turanská, M; Vidaki, A; Zatkalíková, L; Ballantyne, J

2012-11-17

380

MicroRNA mediates DNA demethylation events triggered by retinoic acid during neuroblastoma cell differentiation.  

PubMed

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation. PMID:20841484

Das, Sudipto; Foley, Niamh; Bryan, Kenneth; Watters, Karen M; Bray, Isabella; Murphy, Derek M; Buckley, Patrick G; Stallings, Raymond L

2010-09-14

381

Structure-activity relationship of polypyridyl ruthenium(II) complexes as DNA intercalators, DNA photocleavage reagents, and DNA topoisomerase and RNA polymerase inhibitors.  

PubMed

To investigate the relationship between the molecular structure and biological activity of polypyridyl Ru(II) complexes, such as DNA binding, photocleavage ability, and DNA topoisomerase and RNA polymerase inhibition, six new [Ru(bpy)(2)(dppz)](2+) (bpy=2,2'-bipyridine; dppz=dipyrido[3,2-a:2,',3'-c]phenazine) analogs have been synthesized and characterized by means of (1)H-NMR spectroscopy, mass spectrometry, and elemental analysis. Interestingly, the biological properties of these complexes have been identified to be quite different via a series of experimental methods, such as spectral titration, DNA thermal denaturation, viscosity, and gel electrophoresis. To explain the experimental regularity and reveal the underlying mechanism of biological activity, the properties of energy levels and population of frontier molecular orbitals and excited-state transitions of these complexes have been studied by density-functional theory (DFT) and time-depended DFT (TDDFT) calculations. The results suggest that DNA intercalative ligands with better planarity, greater hydrophobicity, and less steric hindrance are beneficial to the DNA intercalation and enzymatic inhibition of their complexes. PMID:23495154

Chen, Xing; Gao, Feng; Yang, Wei-Yan; Zhou, Zhu-Xin; Lin, Jin-Qiang; Ji, Liang-Nian

2013-03-01

382

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.  

PubMed

A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The 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 (12.9× above background), resulting in an approximately 2- to 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 polymerase chain reactions by measuring enhancement of donor fluorescence on 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

2012-09-19

383

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA  

PubMed Central

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique’s versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing.

Wienken, Christoph J.; Baaske, Philipp; Duhr, Stefan; Braun, Dieter

2011-01-01

384

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

385

Accurate Quantification of Hepatitis C Virus (HCV) RNA from All HCV Genotypes by Using Branched-DNA Technology  

Microsoft Academic Search

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding

JILL DETMER; ROBERT LAGIER; JULIE FLYNN; CRYSTLE ZAYATI; JANICE KOLBERG; MARK COLLINS; MICKEY URDEA; ANDRAY SANCHEZ-PESCADOR

1996-01-01

386

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

PubMed

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

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

2013-09-03

387

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

SciTech Connect

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

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

2010-03-08

388

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor  

PubMed Central

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 Ĺ resolution. The structure demonstrates the remarkable structural conservation of the ?-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.

Bolduc, Jill M.; Spiegel, P. Clint; Chatterjee, Piyali; Brady, Kristina L.; Downing, Maureen E.; Caprara, Mark G.; Waring, Richard B.; Stoddard, Barry L.

2003-01-01

389

tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma.  

PubMed

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles. PMID:23297232

Maute, Roy L; Schneider, Christof; Sumazin, Pavel; Holmes, Antony; Califano, Andrea; Basso, Katia; Dalla-Favera, Riccardo

2013-01-07

390

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

391

Discovery of Widespread GTP-Binding Motifs in Genomic DNA and RNA  

PubMed Central

SUMMARY Biological RNAs that bind small-molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genomeencoded RNA fragments for naturally occurring GTP aptamers. Several classes of aptamers were identified, including one ("the G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ?75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (?300 µM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

Curtis, Edward A.; Liu, David R.

2013-01-01

392

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD  

SciTech Connect

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of {alpha}-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.

Nowotny, Marcin; Cerritelli, Susana M.; Ghirlando, Rodolfo; Gaidamakov, Sergei A.; Crouch, Robert J.; Yang, Wei (NIH)

2008-07-09

393

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

PubMed Central

The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1–2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000–2000 nucleotides downstream (5? ? 3?) of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5?-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.

Morimatsu, Katsumi; Wu, Yun; Kowalczykowski, Stephen C.

2012-01-01

394

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

PubMed

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

2013-05-14

395

H-NS can facilitate specific DNA-binding by RNA polymerase in AT-rich gene regulatory regions.  

PubMed

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

Singh, Shivani S; Grainger, David C

2013-06-20

396

MicroRNA regulation via DNA methylation during the morula to blastocyst transition in mice.  

PubMed

Epigenetic regulation is responsible for transcriptional silencing of genes and parental imprinting. This study addresses the question whether microRNAs (miRNAs) could be affected by DNA methylation during morula-blastocyst transition. Mouse embryos were treated with/without a DNA methyltransferase inhibitor (5-aza-2'-deoxycytidine, 5-aza-dC, 10 nM-5 ?M). Changes of miRNAs were analyzed by quantitative real-time (Q-PCR)-based megaplex pre-amp microRNA assays. Development from morula to blastocyst in mice was inhibited by 5-aza-dC in a dose-dependent manner (10 nM-5 ?M), with half of the embryos arrested at morula stage when treated with levels of 5-aza-dC as low as 50 nM. In total, 48 down-regulated microRNAs and 17 up-regulated microRNAs (?5-fold changes) were identified after 5-aza-dC treatment, including let-7e, mir-20a, mir-21, mir-34b, mir-128b and mir-452. Their predicted targets were selected based on software analysis, published databases and further confirmed by Q-PCR. At least eight targets, including dnmt3a, jagged 1, sp1, edg2, abcg4, numa1, tmsb10 and csf1r were confirmed. In conclusion, 5-aza-dC-modified microRNA profiles and identification of the microRNA's targets during the morula to blastocyst stage in mice provide information that helps us to explore the relationship between fertility, microRNA regulation and epigenetic intervention. PMID:22053057

Lee, Yee-Ming; Chen, Huei-Wen; Maurya, Pawan Kumar; Su, Ching-Mao; Tzeng, Chii-Ruey

2011-11-03

397

An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma.  

PubMed Central

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.

Kern, D; Collins, M; Fultz, T; Detmer, J; Hamren, S; Peterkin, J J; Sheridan, P; Urdea, M; White, R; Yeghiazarian, T; Todd, J

1996-01-01

398

DUPLEX: A molecular mechanics program in torsion angle space for computing structures of DNA and RNA  

SciTech Connect

DUPLEX produces energy minimized structures of DNA and RNA of any base sequence for single and double strands. The smallest subunits are deoxydinucleoside monophosphates, and up to 12 residues, single or double stranded can be treated. In addition, it can incorporate NMR derived interproton distances an constraints in the minimizations. Both upper and lower bounds for these distances can be specified. The program has been designed to run on a UNICOS Cray supercomputer, but should run, albeit slowly, on a laboratory computer such as a VAX or a workstation.

Hingerty, B.E.

1992-07-01

399

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

400

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.  

PubMed Central

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents. Images

Taylor, N R; Kaplan, B E; Swiderski, P; Li, H; Rossi, J J

1992-01-01

401

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

PubMed Central

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

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

1996-01-01

402

Effects of Delay in the Snap Freezing of Colorectal Cancer Tissues on the Quality of DNA and RNA  

PubMed Central

Purpose The success of basic molecular research using biospecimens strongly depends on the quality of the specimen. In this study, we evaluated the effects of delayed freezing time on the stability of DNA and RNA in fresh frozen tissue from patients with colorectal cancer. Methods Tissues were frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A260/A280) and agarose gel electrophoresis were evaluated. In addition, the RNA integrity number (RIN) was assayed for the analysis of the RNA integrity. Results Regardless of delayed freezing time, all DNA and RNA samples revealed A260/A280 ratios of more than 1.9, and all DNA samples showed a discrete, high-molecular-weight band on agarose gel electrophoresis. The RINs were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and 4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though the concentration of RNA was not affected by delayed freezing, the RNA integrity was decreased with increasing delayed freezing time. Conclusion According to the RIN results, we recommend that the collection of colorectal cancer tissue should be done within 10 minutes for studies requiring RNA of high quality and within 30 minutes for usual RNA studies.

Hong, Sang Hyun; Baek, Hyun Ah; Jang, Kyu Yun; Chung, Myoung Ja; Moon, Woo Sung; Kang, Myoung Jae; Lee, Dong Geun

2010-01-01

403

Improvements of the membrane filter method for DNA:rRNA hybridization  

Microsoft Academic Search

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was\\u000a to avoid DNA release from filter discs during hybridization.\\u000a \\u000a \\u000a 1. \\u000a \\u000a Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.\\u000a \\u000a \\u000a \\u000a \\u000a 2. \\u000a \\u000a Duplexing is over in 8–10 hr.\\u000a \\u000a \\u000a \\u000a \\u000a 3. \\u000a \\u000a Formamide has to be very pure

J. De Ley; J. De Smedt

1975-01-01

404

MicroRNA and DNA Methylation Alterations Mediating Retinoic Acid Induced Neuroblastoma Cell Differentiation  

PubMed Central

Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methytransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

Stallings, Raymond L; Foley, Niamh H; Bray, Isabella M; Das, Sudipto; Buckley, Patrick G

2011-01-01

405

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

PubMed

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

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

2013-06-09

406

Cell-specific organization of the 5S ribosomal RNA gene cluster DNA loop domains in spermatozoa and somatic cells.  

PubMed

DNA in eucaryotic cells is organized into loop domains, ranging in size from 25 to 100 kb, that are attached at their bases to the structural component of the nucleus termed the nuclear matrix. These DNA loop domains have been shown to be important in the regulation of both DNA replication and RNA transcription. In this study we have compared the structural organization of the DNA loop domains of the 5S rRNA gene cluster in sperm, liver, and brain nuclei in the Syrian golden hamster. The individual loop domains were visualized by fluorescent in situ hybridization to protamine (sperm)- and histone (somatic)-depleted nuclei, termed nuclear matrix halo preparations. We found that in sperm nuclei, the 5S rRNA gene cluster was organized into three small loop domains that were approximately 48 kb each. In both types of somatic cell nuclei examined, the 5S rRNA gene cluster was or