Sample records for macromolecules dna rna

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

    PubMed Central

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

    2010-01-01

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

  2. Molecular Biology DNA: The Genetic Macromolecule

    E-print Network

    of the E.coli genome. The experiment will allow the student to indirectly observe the central dogma DNA molecule) to a bacterial strain of E. coli called HB101. The plasmid has been engineered supports growth of E.coli. Some plates will also contain ampicillin, a potent antibiotic. Some plates

  3. Fabricating RNA Microarrays with RNA-DNA Surface Ligation Chemistry

    E-print Network

    Fabricating RNA Microarrays with RNA-DNA Surface Ligation Chemistry Hye Jin Lee, Alastair W. Wark to create renewable RNA microarrays from single-stranded DNA (ssDNA) microar- rays on gold surfaces of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array

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

    NASA Astrophysics Data System (ADS)

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

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

  5. Quantum Confinement in Hydrogen Bond of DNA and RNA

    E-print Network

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

    2015-02-09

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

  6. Quantum Confinement in Hydrogen Bond of DNA and RNA

    E-print Network

    Santos, da Silva dos; Ricotta, Regina Maria

    2015-01-01

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

  7. RNA-templated DNA origami structures.

    PubMed

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

    2013-04-11

    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

  8. RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates.

    PubMed

    Hase, Katsunori; Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Wada, Keiji; Kabuta, Tomohiro

    2015-07-27

    Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA. PMID:26038313

  9. RNA-directed DNA methylation in Arabidopsis

    PubMed Central

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

    2002-01-01

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

  10. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    PubMed Central

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  11. 1 Central Dogma. DNA RNA (transcription)

    E-print Network

    1 Central Dogma. DNA RNA (transcription) (translation) . #12 Gene/protein name identification Gene/protein/drug relation extraction (activation, suppression) Disease-related gene/protein identification Information Extractor Gene/protein name identification Gene/protein

  12. Interaction of Sulforaphane with DNA and RNA

    PubMed Central

    Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

    2015-01-01

    Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

  13. An energetic model for macromolecules unraveling

    E-print Network

    De Tommasi, Domenico; Puglisi, Giuseppe; Saccomandi, Giuseppe

    2013-01-01

    We propose a simple approach, based on the minimization of the total (entropic plus unfolding) energy of a two-state system, describing the stretch-induced unfolding of macromolecules (proteins, silks, nanopolymers, DNA/RNA). The model is fully analytical and enlightens the role of the different energetic components regulating the unfolding evolution. As an explicit example of application we compare the analytical results with the titin Atomic Force Microscopy experiments showing the ability of the model to quantitatively reproduce the mechanical behavior of macromolecules unfolding.

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

    PubMed Central

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

    2009-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2007-03-01

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

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

    PubMed Central

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

    2008-01-01

    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. PMID:19693406

  17. International Journal of Biological Macromolecules 41 (2007) 251259 Investigations on CH interactions in RNA binding proteins

    E-print Network

    Babu, M. Madan

    2007-01-01

    . There was an average of 55 C­H· · · interactions per protein and also there was an average of one significant C­H· · · interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C­H· · · interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution

  18. Properties of Macromolecules

    NSDL National Science Digital Library

    Keirle, Matt

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

  19. Nuclear Overhauser effect in specifically deuterated macromolecules: NMR assay for unusual base pairing in transfer RNA.

    PubMed Central

    Sánchez, V; Redfield, A G; Johnston, P D; Tropp, J

    1980-01-01

    We demonstrate a fairly general method for identification of NMR absorption lines of macromolecues extracted from microorganisms, based on nuclear Overhauser effects (NOE). Several NOE in tRNA are observable between resolved imino proton resonances and ring carbon resonances that are either C(2) protons of adenine or C(8) protons of adenine or guanine. Yeast tRNAPhe was deuterated at the purine C(8) positins by heating in 2H2O and also biosynthetically. NOE between imino protons and adenine C(2) protons of standard A . U base pairs would not be affected by such a label, but some other NOE that might be otherwise similar, such as those of reverse Hoogsteen base pairs, should disappear. Six NOE were shown to be from standard A . U pairs by their nondisappearance. Four NOE from methyl resonances to aromatic proton resonances did disappear. The results disagree with previous assignments based on ring-current theories of imino proton NMR shifts. PMID:7003592

  20. Trivalent lanthanide ions do not cleave RNA in DNA-RNA hybrids

    SciTech Connect

    Kolasa, K.A.; Morrow, J.R.; Sharma, A.P. (State Univ. of New York, Buffalo, NY (United States))

    1993-09-15

    Lanthanide(III) complexes rapidly catalyze cleavage of single-stranded RNA. RNA cleavage by lanthanide complexes is, however, dependent on RNA structure. A DNA-RNA hybrid formed by annealing a complementary oligodeoxynucleotide to t-RNA[sup phe] is found to be inert to cleavage by a europium(III) hexadentate Schiff base complex and by Eu(CO[sub 2]CH[sub 3])[sub 3]. Because DNA-RNA hybrids are important structures in antisense oligonucleotide strategies, these results may influence the design of antisense oligonucleotides with attached metal complex cleaving agents.

  1. Archived Formalin-Fixed Paraffin-Embedded (FFPE) Blocks: A Valuable Underexploited Resource for Extraction of DNA, RNA, and Protein

    PubMed Central

    Patel, Miral S.; McGarvey, Diane; LiVolsi, Virginia A.; Baloch, Zubair W.

    2013-01-01

    Formalin-fixed paraffin-embedded (FFPE) material presents a readily available resource in the study of various biomarkers. There has been interest in whether the storage period has significant effect on the extracted macromolecules. Thus, in this study, we investigated if the storage period had an effect on the quantity/quality of the extracted nucleic acids and proteins. We systematically examined the quality/quantity of genomic DNA, total RNA, and total protein in the FFPE blocks of malignant tumors of lung, thyroid, and salivary gland that had been stored over several years. We show that there is no significant difference between macromolecules extracted from blocks stored over 11–12 years, 5–7 years, or 1–2 years in comparison to the current year blocks. PMID:24845430

  2. MicroRNA mediates DNA methylation of target genes.

    PubMed

    Hu, Wangxiong; Wang, Tingzhang; Xu, Jianhong; Li, Hongzhi

    2014-02-21

    Small RNAs represented by microRNA (miRNA) plays important roles in plant development and responds to biotic and abiotic stresses. Previous studies have placed special emphasis on gene-repression mediated by miRNA. In this work, the DNA methylation pattern of microRNA genes (MIRs) was interrogated. Full-length cDNA and EST were used to confirm the entity of pri-miRNA. In parallel, miRNA in 24 nucleotides (nt) was pooled to detect chromatin modification effect by using bisulfite sequencing data. 97 MIRs were supported by full-length cDNA and 30 more were hit by EST. Notably, methylation levels of conserved MIRs were significantly lower than the non-conserved at all contexts (CG, CHG, and CHH). Additionally, a substantial part of 24-nt miRNA was able to induce target site methylation, providing a broader perspective for researchers. PMID:24508262

  3. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    PubMed Central

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

    2014-01-01

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

  4. An absolute requirement for serum macromolecules in phytohaemagglutinin-induced human lymphocyte DNA synthesis.

    PubMed Central

    Yachnin, S; Raymond, J

    1975-01-01

    We have examined the effect of different variables such as tissue culture media, with or without various supplements, lymphocyte isolation techniques, lymphocyte contamination by autologous red blood cells and platelets, and lymphocyte numbers, on the requirement for serum during phytohaemagglutinin (PHA) induced DNA synthesis in human lymphocytes. At all mitogen doses tested, we have found that dialysable constituents of serum enrich the ability of all tissue culture media to support lymphocyte DNA synthesis; however, human lymphocytes display an absolute requirement for nondialysable macromolecular constituents of serum in order to synthesize DNA. PMID:1212814

  5. Characterization of the 3? exonuclease of Chlamydophila pneumoniae endonuclease IV on double-stranded DNA and the RNA strand of RNA\\/DNA hybrid

    Microsoft Academic Search

    Xipeng Liu; Yang Zhang; Rubing Liang; Jingli Hou; Jianhua Liu

    2007-01-01

    Endonuclease IV has AP endonuclease and 3?-repair diesterase activities. Here, we report Chlamydophila pneumoniae endonuclease IV (CpEndoIV) could hydrolyze the ds DNA and the RNA strand of RNA\\/DNA hybrid from the 3? end, yet the DNA strand of RNA\\/DNA hybrid was not the effective substrate of CpEndoIV. The optimal pH for 3? exonuclease on double-stranded (ds) DNA and RNA\\/DNA hybrids

  6. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maci?g-Dorszy?ska, Monika; W?grzyn, Grzegorz; Guzow-Krzemi?ska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus. PMID:24571086

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

    PubMed

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

    2014-11-01

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

  8. Chemical methods of DNA and RNA fluorescent labeling.

    PubMed Central

    Proudnikov, D; Mirzabekov, A

    1996-01-01

    Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips. PMID:8948646

  9. Inhibition of HIV-1 proviral DNA synthesis and RNA accumulation by mismatched dsRNA.

    PubMed

    Montefiori, D C; Pellegrino, M G; Robinson, W E; Engle, K; Field, M; Mitchell, W M; Gillespie, D H

    1989-02-15

    The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding. PMID:2784055

  10. Observation of Single-Protein and DNA Macromolecule Collisions on Ultramicroelectrodes.

    PubMed

    Dick, Jeffrey E; Renault, Christophe; Bard, Allen J

    2015-07-01

    Single-molecule detection is the ultimate sensitivity in analytical chemistry and has been largely unavailable in electrochemical analysis. Here, we demonstrate the feasibility of detecting electrochemically inactive single biomacromolecules, such as enzymes, antibodies, and DNA, by blocking a solution redox reaction when molecules adsorb and block electrode sites. By oxidizing a large concentration of potassium ferrocyanide on an ultramicroelectrode (UME, radius ?150 nm), time-resolved, discrete adsorption events of antibodies, enzymes, DNA, and polystyrene nanospheres can be differentiated from the background by their "footprint". Further, by assuming that the mass transport of proteins to the electrode surface is controlled mainly by diffusion, a size estimate using the Stokes-Einstein relationship shows good agreement of electrochemical data with known protein sizes. PMID:26108405

  11. Birefringence of macromolecules. Wiener's theory revisited, with applications to DNA and tobacco mosaic virus.

    PubMed Central

    Oldenbourg, R; Ruiz, T

    1989-01-01

    We summarize Wiener's theory of the dielectric constant of heterogeneous systems and extend its application to suspensions of particles with corrugated surfaces and interstitial solvent. We retain a simple geometrical shape for the particles and account specifically for the solvent associated with the particles. We calculate the birefringence of the rodshaped Tobacco Mosaic Virus (TMV) particle and of DNA and find excellent agreement between our numerical results and experimental values from the literature. PMID:2752088

  12. Lipoplexes versus nanoparticles: pDNA/siRNA delivery.

    PubMed

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

    2013-02-01

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

  13. Localization of DNA and RNA in Eosinophil Secretory Granules

    Microsoft Academic Search

    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

    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

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

    PubMed

    Rambo, Robert P; Tainer, John A

    2010-02-01

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

  15. qiRNA, a new type of small interfering RNA induced by DNA damage

    PubMed Central

    Lee, Heng-Chi; Chang, Shwu-Shin; Choudhary, Swati; Aalto, Antti P.; Maiti, Mekhala; Bamford, Dennis H.; Liu, Yi

    2010-01-01

    Summary RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNA) and microRNAs, several types of endogenously produced small RNAs play important roles in gene regulation, germ cell maintenance and transposon silencing 1–4. Production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases (RdRPs) to make double stranded RNA (dsRNA). The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a novel class of small RNAs in the filamentous fungus Neurospora. This class of small RNAs, named qiRNAs for their association with QDE-2, are about 20–21 nt long (several nt shorter than Neurospora siRNAs) with a strong preference for uridine at the 5? end and originate mostly from the ribosomal DNA locus. Production of qiRNAs requires the RdRP QDE-1, the Werner/Bloom RecQ DNA helicase homolog QDE-3 and dicers. qiRNA biogenesis also requires DNA damage-induced aRNAs as precursor, a process that is dependent on QDE-1 and QDE-3. Surprisingly, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. In addition, the Neurospora RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs in DNA damage response by inhibiting protein translation. PMID:19444217

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

    E-print Network

    Bass, Brenda L.

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

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

    PubMed

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

    2012-04-01

    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

  18. Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake †

    PubMed Central

    Robarts, Richard D.; Wicks, Richard J.; Sephton, Lynne M.

    1986-01-01

    The incorporation of [methyl-3H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-3H]thymidine studies of aquatic bacterial production. PMID:16347241

  19. The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

    PubMed Central

    Yang, Qiuying; Chang, Shwu-Shin; Huang, Guocun; Fisher, Daniel; Cha, Joonseok; Poranen, Minna M.; Bamford, Dennis H.; Liu, Yi

    2010-01-01

    The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways. PMID:20957187

  20. Fluorescent in situ hybridization of mitochondrial DNA and RNA.

    PubMed

    Alán, Lukáš; Zelenka, Jaroslav; Ježek, Jan; Dlasková, Andrea; Ježek, Petr

    2010-01-01

    To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization. DNase I spread the signal over mitochondrial tubules. Future applications are discussed. PMID:21125028

  1. ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid.

    PubMed

    Birrento, Monica L; Bryan, Tracy M; Samosorn, Siritron; Beck, Jennifer L

    2015-07-01

    Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand (1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism. Graphical Abstract ?. PMID:25906017

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2013-01-01

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

  4. Induction of fos RNA by DNA-damaging agents

    SciTech Connect

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

    1989-04-01

    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.

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

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

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

  6. Characterization of marine prokaryotic communities via DNA and RNA

    Microsoft Academic Search

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

    1994-01-01

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

  7. Molecular-Sized DNA or RNA Sequencing Machine

    Cancer.gov

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

  8. Analyses of DNA, RNA and Protein

    E-print Network

    Dellaire, Graham

    ;Hybridization technology #12;Molecular techniques for Analyzing DNA #12;Southern blotting #12;Detection of gene deletion by Southern blotting #12; Southern blotting can be used to detect large alterations of PCR over Southern blotting Fast Sensitive Inexpensive #12; PCR can be used to screen for unknown

  9. RNA Isolation -cDNA Procedures Precautions -

    E-print Network

    Cruzan, Mitchell B.

    Sodium Citrate plus 1.2 M NaCl) - 60% Ethanol - Sodium citrate storage buffer (1 mM, pH 6.4) Procedure: 1. - Centrifuge @ 9,000 rpm for 15 minutes. #12;4. RNA precipitation. - Transfer aqueous (upper) layer (2 x 200 :L solution: - Add 50 :L of Sodium Citrate storage buffer per sample. - Add RNAsecure reagent to dilute to 1X

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2006-01-01

    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

  12. FUS-regulated RNA metabolism and DNA damage repair

    PubMed Central

    Zhou, Yueqin; Liu, Songyan; Öztürk, Arzu; Hicks, Geoffrey G

    2014-01-01

    Cytoplasmic inclusion of RNA binding protein FUS/TLS in neurons and glial cells is a characteristic pathology of a subgroup of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dysregulation of RNA metabolism caused by FUS cytoplasmic inclusion emerges to be a key event in FUS-associated ALS/FTD pathogenesis. Our recent discovery of a FUS autoregulatory mechanism and its dysregulation in ALS-FUS mutants demonstrated that dysregulated alternative splicing can directly exacerbate the pathological FUS accumulation. We show here that FUS targets RNA for pre-mRNA alternative splicing and for the processing of long intron-containing transcripts, and that these targets are enriched for genes in neurogenesis and gene expression regulation. We also identify that FUS RNA targets are enriched for genes in the DNA damage response pathway. Together, the data support a model in which dysregulated RNA metabolism and DNA damage repair together may render neurons more vulnerable and accelerate neurodegeneration in ALS and FTD. PMID:25083344

  13. Characterization of a Viroid-Derived RNA Promoter for the DNA-Dependent RNA Polymerase from Escherichia coli

    E-print Network

    Perreault, Jean-Pierre

    three viroids form the avsunviroidae group. Avocado sunblotch viroid (ASBVd) is believed to adoptCharacterization of a Viroid-Derived RNA Promoter for the DNA-Dependent RNA Polymerase from-cleaving hammerhead RNA sequences found in RNA derived from the peach latent mosaic viroid (PLMVd). The features

  14. Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells

    Microsoft Academic Search

    Lydia V Rump; Benedicta Asamoah; Narjol Gonzalez-Escalona

    2010-01-01

    BACKGROUND: The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets invA mRNA for the detection of live Salmonella cells. The assay of

  15. Single-Molecule Electrical Random Resequencing of DNA and RNA

    PubMed Central

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

    2012-01-01

    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

  16. Single-Molecule Electrical Random Resequencing of DNA and RNA

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    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.

  17. Excited states of protonated DNA/RNA bases Matias Berdakin1

    E-print Network

    Boyer, Edmond

    is critical since their photochemistry constitutes a fundamental step in radiation-induced DNA/RNA damage1 Excited states of protonated DNA/RNA bases Matias Berdakin1 , Géraldine Féraud2 , Claude Dedonder of the excited states to the ground state in DNA/RNA bases is a necessary process to ensure the photostability

  18. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation.

    PubMed

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R; Pradhan, Sriharsa

    2015-07-13

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

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

    PubMed Central

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

    2014-01-01

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

  20. DNA and RNA synthesis by postpubertal undescended testis in vitro

    SciTech Connect

    Nakamura, M.; Nonomura, N.; Namiki, M.; Okuyama, A.; Koh, E.; Kondoh, N.; Fujioka, H.; Nishimune, Y.; Matsumoto, K.; Matsuda, M.

    1989-01-01

    To study the spermatogenic potential of postpubertal undescended testis, the /sup 3/H-thymidine and /sup 14/C-uridine incorporations into normally descended and inguinal undescended testes were examined. The results suggest that DNA synthesis by the inguinal undescended testis is remarkably inhibited but, on the contrary, RNA synthesis is not. The cell-proliferative ability of postpubertal undescended testis may be impaired in spite of the retention of cell viability in the testis.

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

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

    2012-01-01

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

  3. Oxidatively generated DNA/RNA damage in psychological stress states.

    PubMed

    Jørgensen, Anders

    2013-07-01

    Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8-oxodG and 8-oxoGuo, respectively). The main hypothesis was that psychological stress states are associated with increased DNA/RNA damage from oxidation. In a study of 40 schizophrenia patients and 40 healthy controls matched for age and gender, we found that 8-oxodG/8-oxoGuo excretion was increased in schizophrenia patients, providing a possible molecular link between schizophrenia and its associated signs of accelerated aging. We found no association between psychopathology, perceived stress or cortisol secretion and 8-oxodG/8-oxoGuo excretion in the patients. In the controls, there were positive correlations between 8-oxodG/8-ocoGuo excretion and 9AM plasma cortisol, but no associations to perceived stress. In an animal study of experimentally induced chronic stress performed in metabolism cages, we found no increase in urinary 8-oxodG/8-oxoGuo or cerebral (hippocampal and frontal cortex) levels of oxidatively generated nucleic acid damage. However, there was a trend towards an increased expression of genes involved in DNA repair, possibly reflecting a compensatory mechanism. In a study of 220 elderly, mostly healthy individuals from the Italian InChianti cohort, we found a significant association between the 24 h urinary cortisol excretion and the excretion of 8-oxodG/8-oxoGuo, determined in the same samples. Collectively, the studies could not confirm an association between psychological stress and oxidative stress on nucleic acids. Systemic oxidatively generated DNA/RNA damage was increased in schizophrenia, and linked to cortisol levels in healthy humans. Finally, the cerebral repair of DNA may be an aspect of the adaptation that, to our knowledge, has not previously been addressed. PMID:23809980

  4. The prebiotic evolutionary advantage of transferring genetic information from RNA to DNA

    PubMed Central

    Leu, Kevin; Obermayer, Benedikt; Rajamani, Sudha; Gerland, Ulrich; Chen, Irene A.

    2011-01-01

    In the early ‘RNA world’ stage of life, RNA stored genetic information and catalyzed chemical reactions. However, the RNA world eventually gave rise to the DNA–RNA–protein world, and this transition included the ‘genetic takeover’ of information storage by DNA. We investigated evolutionary advantages for using DNA as the genetic material. The error rate of replication imposes a fundamental limit on the amount of information that can be stored in the genome, as mutations degrade information. We compared misincorporation rates of RNA and DNA in experimental non-enzymatic polymerization and calculated the lowest possible error rates from a thermodynamic model. Both analyses found that RNA replication was intrinsically error-prone compared to DNA, suggesting that total genomic information could increase after the transition to DNA. Analysis of the transitional RNA/DNA hybrid duplexes showed that copying RNA into DNA had similar fidelity to RNA replication, so information could be maintained during the genetic takeover. However, copying DNA into RNA was very error-prone, suggesting that attempts to return to the RNA world would result in a considerable loss of information. Therefore, the genetic takeover may have been driven by a combination of increased chemical stability, increased genome size and irreversibility. PMID:21724606

  5. Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

    SciTech Connect

    Mao, Xun; Liu, Guodong; Wang, Shengfu; Lin, Yuehe; Zhang, Aiguo; Zhang, Lurong; Ma, Yunqing

    2008-11-01

    We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

  6. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products.

    PubMed

    Fleming, Aaron M; Alshykhly, Omar; Zhu, Judy; Muller, James G; Burrows, Cynthia J

    2015-06-15

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design. PMID:25853314

  7. Dynamics of Biological Macromolecules: Not a Simple Slaving by Hydration Water

    SciTech Connect

    Kisliuk, Alexander [ORNL; Sokolov, Alexei P [ORNL; Mamontov, Eugene [ORNL; Khodadadi, S [University of Akron; Roh, J H [University of Maryland; Tyagi, M. [NCNR and University of Maryland; Briber, R M [University of Maryland; Woodson, S.A. [Johns Hopkins University

    2010-01-01

    We studied the dynamics of hydrated tRNA using neutron and dielectric spectroscopy techniques. A comparison of our results with earlier data reveals that the dynamics of hydrated tRNA is slower and varies more strongly with temperature than the dynamics of hydrated proteins. At the same time, tRNA appears to have faster dynamics than DNA. We demonstrate that a similar difference appears in the dynamics of hydration water for these biomolecules. The results and analysis contradict the traditional view of slaved dynamics, which assumes that the dynamics of biological macromolecules just follows the dynamics of hydration water. Our results demonstrate that the dynamics of biological macromolecules and their hydration water depends strongly on the chemical and three-dimensional structures of the biomolecules. We conclude that the whole concept of slaving dynamics should be reconsidered, and that the mutual influence of biomolecules and their hydration water must be taken into account.

  8. NUCLEIC ACID CATALYSTS: COMPARING THE MECHANISMS OF DNA AND RNA ENZYMES

    Microsoft Academic Search

    Kinjal Desai; Nikita Ekhelikar; Jennifer Kasbohm; Parth Kothari

    Based on previous research on DNA enzyme catalysis, it has been shown that divalent ions are effective in aiding in the cleavage of RNA substrate by the 10-23 DNA enzyme. In previous work by other laboratories, the 10-23 DNA enzyme was isolated by in vitro evolution and has been shown to cleave RNA effectively. It does this by binding to

  9. Thermodynamic dependence of DNA\\/DNA and DNA\\/RNA hybridization reactions on temperature and ionic strength

    Microsoft Academic Search

    Brian E. Lang; Frederick P. Schwarz

    2007-01-01

    The thermodynamics of 5?-ATGCTGATGC-3? binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, ?G° of the DNA hybridization reactions increased by about 6 kJ mol?1 from 20 °C to 37 °C and exhibited heat capacity changes of ?1.42±0.09 kJ mol?1 K?1

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

    PubMed

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

    2015-03-01

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

  11. Human U1 RNA pseudogenes may be generated by both DNA- and RNA-mediated mechanisms.

    PubMed Central

    Denison, R A; Weiner, A M

    1982-01-01

    Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event. Images PMID:6085956

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

    PubMed

    Seligmann, Hervé

    2013-09-01

    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

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

    Microsoft Academic Search

    1982-01-01

    The double strands formed by the RNA, DNA, and RNA.DNA hybrid oligonucleotides rCAâG + rCUâG, dCAâG + dCtâG and rCAâG + dCTâG were studied in order to determine the differences in the stability and dynamics of RNA and DNA. The thermodynamics of double-strand formation were determined by measuring the absorbance vs temperature at 260 nm for different strand concentrations. The

  14. The in Vivo Binding of ß-Propiolactoneto Mouse Skin DNA, RNA, and Protein1

    Microsoft Academic Search

    N. H. Colburn; R. K. Boutwell

    SUMMARY The binding of tritium-labeled \\/3-propiolactone to mouse skin DNA, RNA, and protein was investigated. Binding of the lactone to RNA and protein, as well as to DNA, was observed. When propiolactone dose or mouse susceptibility was varied, the binding to skin DNA, RNA, and protein was found to correlate with initiation of tumorigenesis. The maximum bind ing of \\/3-propiolactone-3H

  15. pubs.acs.org/Macromolecules Published on Web 11/02/2009 r 2009 American Chemical Society 9170 Macromolecules 2009, 42, 91709182

    E-print Network

    Shaqfeh, Eric

    pubs.acs.org/Macromolecules Published on Web 11/02/2009 r 2009 American Chemical Society 9170 Macromolecules 2009, 42, 9170­9182 DOI: 10.1021/ma901348j Experimental and Numerical Studies of Tethered DNA

  16. Deploying RNA and DNA with Functionalized Carbon Nanotubes.

    PubMed

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

    2013-03-21

    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

  17. Biological Macromolecule Crystallization Database

    National Institute of Standards and Technology Data Gateway

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

  18. Endogenous RNA-Directed DNA Polymerase Activity in Uninfected Chicken Embryos

    Microsoft Academic Search

    Chil-Yong Kang; Howard M. Temin

    1972-01-01

    Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA

  19. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.

    PubMed

    Jiang, Fuguo; Zhou, Kaihong; Ma, Linlin; Gressel, Saskia; Doudna, Jennifer A

    2015-06-26

    Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes. PMID:26113724

  20. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-29

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

  3. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    NASA Astrophysics Data System (ADS)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

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

    PubMed Central

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

    2014-01-01

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

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

    Microsoft Academic Search

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

    1985-01-01

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

  6. DNA-RNA hybrids and transcriptional activity in Chironomus polytene chromosomes

    Microsoft Academic Search

    J. L. Díez; D. Barettino

    1984-01-01

    The distribution of DNA-RNA hybrids was studied in fixed Chironomus polytene chromosomes by means of specific antibodies directed against DNA-RNA hybrids. Attention was mainly focused on the relationship between detection of hybrids and local transcriptional activity. As a model to test such a relationship Balbiani rings, whose transcriptional activity was experimentally modified, and a new set of puffs induced by

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

    E-print Network

    Kreil, David

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

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

    E-print Network

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

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

    PubMed Central

    Peña-Llopis, Samuel; Brugarolas, James

    2014-01-01

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

  10. A human RNA polymerase II complex associated with SRB and DNA-repair proteins

    Microsoft Academic Search

    Edio Maldonado; Ramin Shiekhattar; Michael Sheldon; Helen Cho; Ronny Drapkin; Paula Rickert; Emma Lees; Carl W. Anderson; Stuart Linn; Danny Reinberg

    1996-01-01

    WE report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins1-3. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.

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

    E-print Network

    Park, Jong-Sang

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

  12. RNA?DNA hybrids initiate quasi-palindrome-associated mutations in highly transcribed yeast DNA.

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2013-01-01

    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

  14. Detection of Genomic DNA Fragmentation during Apoptosis (DNA Ladder) and the Simultaneous Isolation of RNA from Low Cell Numbers

    Microsoft Academic Search

    Peter T. Daniel; Isrid Sturm; Silke Ritschel; Katrin Friedrich; Bernd Dörken; Peter Bendzko; Timo Hillebrand

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the

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

    PubMed

    Allison, Simon J; Milner, Jo

    2014-01-01

    Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3' end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3'-DNA/ds siRNA and includes a 3'-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3'-DNA/ds siRNA, (II) passenger-3'-DNA/ds siRNA, (III) guide-3'-DNA/ss siRNA, and (IV) passenger-3'-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I-III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3'-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3'-DNA/siRNA constructs are discussed.Molecular Therapy-Nucleic Acids (2014) 2, e141; doi:10.1038/mtna.2013.68; published online 7 January 2014. PMID:24399205

  16. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral microRNA shuttles

    Microsoft Academic Search

    Saket Chattopadhyay; Abdullah Ely; Kristie Bloom; Marc S. Weinberg; Patrick Arbuthnot

    2009-01-01

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of

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

    Microsoft Academic Search

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

    1990-01-01

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

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

    PubMed Central

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

    1993-01-01

    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. PMID:12271056

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

    Microsoft Academic Search

    Toru Fujiwara; Biao Ding; Steven A. Lommel; William J. Lucasai

    1993-01-01

    Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluores- cently 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

  20. Transcription Factor UAF, Expansion and Contraction of Ribosomal DNA (rDNA) Repeats, and RNA Polymerase Switch in Transcription of Yeast rDNA

    Microsoft Academic Search

    MELANIE OAKES; IMRAN SIDDIQI; LOAN VU; JOHN ARIS; MASAYASU NOMURA

    1999-01-01

    Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demon- strated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats.

  1. Antiapoptotic small interfering RNA as potent adjuvant of DNA vaccination in a mouse mammary tumor model.

    PubMed

    Dharmapuri, Sridhar; Aurisicchio, Luigi; Biondo, Antonella; Welsh, Natalie; Ciliberto, Gennaro; La Monica, Nicola

    2009-06-01

    In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines. It results in increased DNA uptake, enhances protein expression, and augments immune responses to the target antigen in a variety of species. To further improve the efficacy of DNA-EP, we evaluated small interfering RNA (siRNA) sequences targeting apoptotic genes as an adjuvant to cancer vaccine. Bak1 or Casp8 siRNA was coadministered with plasmid DNA encoding the extracellular and transmembrane domains of rat HER2 ECD.TM to BALB-neuT mice, which spontaneously develop HER2/neu-positive mammary tumors. The combination regimen significantly reduced spontaneous tumor progression in BALB-neuT mice, in an advanced disease setting, when compared with DNA-EP alone. The antitumor effect was associated with a noteworthy antibody isotype switch from IgG1 to IgG2a, when siRNA was coadministered with DNA-EP. CD8+ T cell responses increased significantly, as did the number of responders to vaccination. Coimmunization of siRNA and DNA-EP at the same physical location was essential for the enhanced therapeutic effect. Silencing of the targeted genes was confirmed by in vitro Western blots. siRNA sequences targeting apoptotic genes Bax and Fas did not improve tumor protection in this mouse model when compared with DNA-EP alone. These data demonstrate that some siRNA sequences can act in concert with DNA-EP to control HER2/neu-positive mammary carcinoma. These observations emphasize the potential of siRNA as adjuvant for therapeutic DNA vaccines. PMID:19222350

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

    SciTech Connect

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

    1985-06-14

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

  3. Single Molecule Photobleaching (SMPB) Technology for Counting of RNA, DNA, Protein and Other Molecules in Nanoparticles and Biological Complexes by TIRF Instrumentation

    PubMed Central

    Zhang, Hui; Guo, Peixuan

    2014-01-01

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The Single Molecule Photobleaching (SMPB) technology for direct counting developed by our team (Shu et al, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. PMID:24440482

  4. Pre-mRNA processing factors meet the DNA damage response

    PubMed Central

    Montecucco, Alessandra; Biamonti, Giuseppe

    2013-01-01

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

  5. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

    PubMed Central

    Pai, Dave A.; Kaplan, Craig D.; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C.; Engelke, David R.

    2014-01-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template. PMID:24614752

  6. Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA

    PubMed Central

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

    1974-01-01

    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 PMID:4139714

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

    PubMed Central

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

    2014-01-01

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

  8. Host defense against DNA virus infection in shrimp is mediated by the siRNA pathway.

    PubMed

    Huang, Tianzhi; Zhang, Xiaobo

    2013-01-01

    The RNA interference (RNAi) system of eukaryotes using siRNAs has been documented as an immune response against invasion by RNA viruses. However, whether the siRNA pathway can be triggered by the infection with DNA viruses in animals remains to be investigated. In the present study, we show that Marsupenaeus japonicus shrimp can generate an antiviral siRNA (vp28-siRNA) in response to infection by a double-stranded DNA virus, white spot syndrome virus (WSSV). After challenging with WSSV, vp28-siRNA is detected in all the WSSV-infected organs and tissues of shrimp as early as 24 h postinfection (p.i.). The results indicate that the host Dicer2 and Ago2 proteins are required for the biogenesis and function of vp28-siRNA, respectively. We show further that vp28-siRNA predominates in the cytoplasm of shrimp hemocytes at 48 h p.i. Knockdown of Dicer2 by special siRNA or inhibition of vp28-siRNA with locked nucleic acid antisense oligonucleotides both lead to a significant increase in WSSV copy number at 24-48 h p.i. Our study highlights a novel aspect of the siRNA pathway in the immune response of animals against infection by DNA viruses. PMID:23065729

  9. Increased systemic oxidatively generated DNA and RNA damage in schizophrenia.

    PubMed

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

    2013-10-30

    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

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

    PubMed Central

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

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

  11. A Simple RNA-DNA Scaffold Templates the Assembly of Monofunctional Virus-Like Particles.

    PubMed

    Garmann, Rees F; Sportsman, Richard; Beren, Christian; Manoharan, Vinothan N; Knobler, Charles M; Gelbart, William M

    2015-06-24

    Using the components of a particularly well-studied plant virus, cowpea chlorotic mottle virus (CCMV), we demonstrate the synthesis of virus-like particles (VLPs) with one end of the packaged RNA extending out of the capsid and into the surrounding solution. This construct breaks the otherwise perfect symmetry of the capsid and provides a straightforward route for monofunctionalizing VLPs using the principles of DNA nanotechnology. It also allows physical manipulation of the packaged RNA, a previously inaccessible part of the viral architecture. Our synthesis does not involve covalent chemistry of any kind; rather, we trigger capsid assembly on a scaffold of viral RNA that is hybridized at one end to a complementary DNA strand. Interaction of CCMV capsid protein with this RNA-DNA template leads to selective packaging of the RNA portion into a well-formed capsid but leaves the hybridized portion poking out of the capsid through a small hole. We show that the nucleic acid protruding from the capsid is capable of binding free DNA strands and DNA-functionalized colloidal particles. Separately, we show that the RNA-DNA scaffold can be used to nucleate virus formation on a DNA-functionalized surface. We believe this self-assembly strategy can be adapted to viruses other than CCMV. PMID:26043403

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

    PubMed Central

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

    2014-01-01

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

  13. 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...

  14. Very Few RNA and DNA Sequence Differences in the Human Transcriptome

    E-print Network

    Hahn, Matthew

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

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

    Microsoft Academic Search

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

    1985-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  17. Targeted Delivery of siRNA-Generating DNA Nanocassettes Using Multifunctional Nanoparticles

    PubMed Central

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

    2013-01-01

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

  18. RNA cell typing and DNA profiling of mixed samples: can cell types and donors be associated?

    PubMed

    Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia

    2013-09-01

    Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling. PMID:23937933

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

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

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

  20. Targeting DNA and RNA in Pathogens: Mode of Action of Amotosalen HCl

    Microsoft Academic Search

    S. Wollowitz

    2004-01-01

    SummaryBlood products for transfusion may contain a wide variety of DNA- and RNA-based pathogens, including those for which there are no current tests, and they are vulnerable to emerging, unknown pathogens, thus compromising the safety of the products [1]. Amotosalen HCl (S-59), in combination with UVA light, has been developed for inactivation of a broad range of DNA- and RNA-based

  1. Effects of Karenia brevis diet on RNA:DNA ratios and egg production of Acartia tonsa

    Microsoft Academic Search

    Christa L. Speekmann; Cammie J. Hyatt; Edward J. Buskey

    2006-01-01

    Karenia brevis is a harmful alga associated with deleterious effects on zooplankton, but the exact cause (e.g. toxin, nutritional inadequacy or starvation) of these adverse effects is not clear. RNA:DNA ratios, fecundity and fecal pellet production of Acartia tonsa were measured on mono-algal and mixed-algal culture diets of K. brevis and Peridinium foliaceum to examine the usefulness of RNA:DNA ratios

  2. E. COLI RNASE HI AND THE PHOSPHONATE-DNA\\/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

    Microsoft Academic Search

    I. Barvík

    2005-01-01

    A model for the complex between E. coli RNase HI and the DNA\\/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3?–O–CH2–P–O–5? or 3?–O–P–CH2–O–5?) on the ability of the modified-DNA\\/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to

  3. Travel Depth, a New Shape Descriptor for Macromolecules: Application to Ligand Binding

    E-print Network

    Sharp, Kim

    Travel Depth, a New Shape Descriptor for Macromolecules: Application to Ligand Binding Ryan G and surface of macromolecules, describing for example the grooves in DNA, the shape of an enzyme active site in a macromolecule, and few computational tools exist to quantify this notion, to visualize it, or analyze

  4. Rat Ribosomal RNA Gene Can Utilize Primate RNA Polymerase I Transcription Machinery: Lack of Absolute Species Specificity in rDNA Transcription

    Microsoft Academic Search

    Asish K. Ghosh; Huifeng Niu; Samson T. Jacob

    1996-01-01

    The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with

  5. DNA polymerase I activity in Escherichia coli is influenced by spot 42 RNA

    SciTech Connect

    Polayes, D.A.; Rice, P.W.; Dahlberg, J.E.

    1988-05-01

    We have shown that the level of DNA polymerase I (Pol I) activity in Escherichia coli in influenced by the level of a 109-nucleotide RNA, spot 42 RNA. Deletion of the gene for spot 42 RNA results in a 20 to 25% decrease in Pol I activity, as assayed by nucleotide incorporation in cell extracts and a decrease in the ability of cells to grow in the presence of the DNA-alkylating agent methyl methanesulfonate. Also, a physiological reduction of the level of spot 42 RNA, by growth in media containing poor carbon sources, results in a corresponding decrease in Pol I activity. Conversely, overproduction of spot 42 RNA results in a 10 to 15% increase in Pol I activity in vitro. Thus, changes in the amount of spot 42 RNA result in relatively small but significant changes in Pol I activity.

  6. Legume genomics: understanding biology through DNA and RNA sequencing

    PubMed Central

    O'Rourke, Jamie A.; Bolon, Yung-Tsi; Bucciarelli, Bruna; Vance, Carroll P.

    2014-01-01

    Background The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. Scope and Conclusions This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes. PMID:24769535

  7. MicroRNA expression profiling and DNA methylation signature for deregulated microRNA in cutaneous T-cell lymphoma.

    PubMed

    Sandoval, Juan; Díaz-Lagares, Angel; Salgado, Rocío; Servitje, Octavio; Climent, Fina; Ortiz-Romero, Pablo L; Pérez-Ferriols, Amparo; Garcia-Muret, Maria P; Estrach, Teresa; Garcia, Mar; Nonell, Lara; Esteller, Manel; Pujol, Ramon M; Espinet, Blanca; Gallardo, Fernando

    2015-04-01

    MicroRNAs usually regulate gene expression negatively, and aberrant expression has been involved in the development of several types of cancers. Microarray profiling of microRNA expression was performed to define a microRNA signature in a series of mycosis fungoides tumor stage (MFt, n=21) and CD30+ primary cutaneous anaplastic large cell lymphoma (CD30+ cALCL, n=11) samples in comparison with inflammatory dermatoses (ID, n=5). Supervised clustering confirmed a distinctive microRNA profile for cutaneous T-cell lymphoma (CTCL) with respect to ID. A 40 microRNA signature was found in MFt including upregulated onco-microRNAs (miR-146a, miR-142-3p/5p, miR-21, miR-181a/b, and miR-155) and downregulated tumor-suppressor microRNAs (miR-200ab/429 cluster, miR-10b, miR-193b, miR-141/200c, and miR-23b/27b). Regarding CD30+ cALCL, 39 differentially expressed microRNAs were identified. Particularly, overexpression of miR-155, miR-21, or miR-142-3p/5p and downregulation of the miR-141/200c clusters were observed. DNA methylation in microRNA gene promoters, as expression regulatory mechanism for deregulated microRNAs, was analyzed using Infinium 450K array and approximately one-third of the differentially expressed microRNAs showed significant DNA methylation differences. Two different microRNA methylation signatures for MFt and CD30+ cALCL were found. Correlation analysis showed an inverse relationship for microRNA promoter methylation and microRNA expression. These results reveal a subgroup-specific epigenetically regulated microRNA signatures for MFt and CD30+ cALCL patients. PMID:25405321

  8. Supplementary Information DNA Targeting Specificity of the RNA-guided Cas9 Nuclease

    E-print Network

    Cai, Long

    Supplementary Information DNA Targeting Specificity of the RNA-guided Cas9 Nuclease Patrick D. Hsu1's recommended protocol. For each well of a 6-well plate, a total of 1 ug of Cas9+sgRNA plasmid was used. For each well of a 24-well plate, a total of 500ng Cas9+sgRNA plasmid was used unless otherwise indicated

  9. Effects of long DNA folding and small RNA stemloop in thermophoresis

    E-print Network

    Tlusty, Tsvi

    with cold fluid around, and also complete isolation from devastating UV solar radiation (4). FollowingEffects of long DNA folding and small RNA stem­loop in thermophoresis Yusuke T. Maedaa,b,c , Tsvi a concen- tration gradient. In this gradient, DNA polymers of different sizes can be separated

  10. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    PubMed Central

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  11. 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

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

    1999-01-01

    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

  12. Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

    PubMed

    Daniel, P T; Sturm, I; Ritschel, S; Friedrich, K; Dörken, B; Bendzko, P; Hillebrand, T

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor. PMID:9887219

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

    Microsoft Academic Search

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

    1997-01-01

    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

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

    Microsoft Academic Search

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

    2000-01-01

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

  15. Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

    PubMed Central

    Kibler-Herzog, L; Zon, G; Uznanski, B; Whittier, G; Wilson, W D

    1991-01-01

    The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations. PMID:1711677

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

    PubMed Central

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

    2014-01-01

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

  17. Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

    PubMed

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

    2015-01-01

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

  18. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function

    PubMed Central

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-01-01

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition–fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates “bifacial polymer nucleic acids” (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure–function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  19. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    PubMed

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  20. Dynamic Monitoring of MicroRNA-DNA Hybridization Using DNAase-Triggered Signal Amplification.

    PubMed

    Qiu, Xiaopei; Liu, Xing; Zhang, Wei; Zhang, Hong; Jiang, Tianlun; Fan, Dongli; Luo, Yang

    2015-06-16

    Dynamically monitoring microRNA (miRNA)-DNA reactions is critical for elucidating various biological processes. However, traditional strategies fail to capture this dynamic event because the original targets are preamplified. In the present study, we developed an amplification-free strategy for real-time monitoring of miRNA-DNA hybridization that integrates the advantages of both duplex-specific nuclease (DSN)-triggered signal amplification and single-stranded DNA probe coating facilitated by reduced graphene oxide. DSN-mediated miRNA recognition was found to consist of two phases: hybridization and hybridization cleavage. In the presence of miRNA and DSN, hybridization of a 22-mer miRNA-DNA could be completed within 7 min by observing the angle increase in a surface plasmon resonance (SPR) biosensor. The subsequent hybridization-cleavage process could be visualized as a gradual SPR angle decrease that occurred until all coated probes were hydrolyzed. In addition, for miRNA-21 detection, the proposed linear signal amplification assay demonstrated a sensitivity of 3 fM over a dynamic range of 5 orders of magnitude. PMID:25962779

  1. DNA mimicry by a high-affinity anti-NF-?B RNA aptamer

    PubMed Central

    Reiter, Nicholas J.; Maher, L. James; Butcher, Samuel E.

    2008-01-01

    The binding of RNA molecules to proteins or other ligands can require extensive RNA folding to create an induced fit. Understanding the generality of this principle involves comparing structures of RNA before and after complex formation. Here we report the NMR solution structure of a 29-nt RNA aptamer whose crystal structure had previously been determined in complex with its transcription factor target, the p502 form of NF-?B. The RNA aptamer internal loop structure has pre-organized features that are also found in the complex, including non-canonical base pairing and cross-strand base stacking. Remarkably, the free RNA aptamer structure possesses a major groove that more closely resembles B-form DNA than RNA. Upon protein binding, changes in RNA structure include the kinking of the internal loop and distortion of the terminal tetraloop. Thus, complex formation involves both pre-formed and induced fit binding interactions. The high affinity of the NF-?B transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA. PMID:18160411

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

    PubMed Central

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

    1996-01-01

    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. PMID:9016675

  3. Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat.

    PubMed

    Sarhan, F; Chevrier, N

    1985-06-01

    Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation. PMID:16664225

  4. The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids.

    PubMed

    Akiyama, Benjamin M; Parks, Joseph W; Stone, Michael D

    2015-06-23

    Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA-DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity. PMID:25940626

  5. The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA–DNA hybrids

    PubMed Central

    Akiyama, Benjamin M.; Parks, Joseph W.; Stone, Michael D.

    2015-01-01

    Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA–DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity. PMID:25940626

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

    PubMed Central

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

    1978-01-01

    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. PMID:77007

  7. Polyelectrolyte and polyampholyte effects in synthetic and biological macromolecules

    E-print Network

    Ngo Minh Toan; Bae-Yeun Ha; D. Thirumalai

    2011-03-09

    The nature of electrostatic interactions involving polyanions modulate the properties of both synthetic and biological macromolecules. Although intensely studied for decades the interplay of many length scales has prevented a complete resolution of some of the basic questions such as how the electrostatic persistence length ($l_e$) varies with ionic strength ($I$). In this review we describe certain characteristics of polyelectrolytes (PAs) and polyampholytes (PAs), which are polymers whose monomers have a random distribution of opposite charges. After reviewing the current theoretical understanding of the dependence of $l_e$ on $I$ we present experimental data that conform to two distinct behavior. For RNA and DNA it is found that that $l_e \\sim I^{-1}$ whereas for some proteins and other polyelectrolytes $l_e \\sim i^{-1/2}$. A scaling type theory, which delineates charge correlation and pure polyelectrolyte effects for the shape of PAs that is valid over a wide range of salt concentration is described. We also use theory and simulations to argue that the distinct stages in the kinetics of collapse of PAs (with a net charge that is small enough to induce globule formation) and PEs (relevant for RNA folding) are similar. In both cases the major initial conformation change involves formation of metastable pearl-necklace structures. In the coarsening process large clusters (pearls) grow at the expense of smaller ones by a process that is reminiscent of Lifshitz-Slyozov mechanism. Finally, recent theories and single molecule experiments on stretching of single stranded DNA and PEs further sheds insights into the complex behavior of charged macromolecules. The survey, which is limited to very few topics, shows the importance of polyelectrolyte effects in a wide range of disciplines.

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

    PubMed

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

    2013-05-01

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

  9. Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on “Click Chemistry”

    PubMed Central

    Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Li, Jiangwei

    2011-01-01

    This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of 3H-uridine incorporation and RNA content provided the means to distinguish quiescent G0 from cycling G1 cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced “click chemistry” approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2?deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry. PMID:21425239

  10. Differential effects of thyroxine on metabolic enzymes and other macromolecules in a freshwater teleost.

    PubMed

    Tripathi, G; Verma, Priyanka

    2003-04-01

    The effects of thyroxine (T(4)) on citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, and protein of various tissues were studied to elucidate the hormonal control of metabolism in a freshwater catfish, Clarias batrachus. T(4) did not produce any significant effect on DNA content of the fish. The CS, RNA, and protein contents of brain, liver, and skeletal muscle of the fish exposed to thiourea for 28 days decreased approximately 50-58% as compared to their levels in control individuals. Injection of T(4) to thiourea-exposed fish produced about three-fold increases in CS, RNA, and protein. These macromolecular inductions by T(4) were blocked by actinomycin D or cycloheximide. This suggests T(4)-induced de novo synthesis of macromolecules and stimulation of aerobic capacity. However, the activities of G6-PDH and LDH of brain, liver, and skeletal muscle of the fish exposed to thiourea increased two times that of the activities in control individuals. Administration of T(4) to thiourea-exposed fish reduced LDH and G6-PDH activities by about 64-74%, which reflects T(4)-dependent inhibition in anaerobic power and selective anabolic activities of the HMP pathway. These differential effects of T(4) on some metabolic enzymes and other important macromolecules may be to meet the other T(4)-induced responses in the freshwater catfish. PMID:12658717

  11. RNA\\/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

    Microsoft Academic Search

    C. Haas; E. Hanson; M. J. Anjos; W. Bär; R. Banemann; A. Berti; E. Borges; C. Bouakaze; A. Carracedo; M. Carvalho; V. Castella; A. Choma; G. De Cock; M. Dötsch; P. Hoff-Olsen; P. Johansen; F. Kohlmeier; P. A. Lindenbergh; B. Ludes; O. Maroñas; D. Moore; M.-L. Morerod; N. Morling; H. Niederstätter; F. Noel; W. Parson; G. Patel; C. Popielarz; E. Salata; P. M. Schneider; T. Sijen; B. Sviežena; M. Turanská; L. Zatkalíková; J. Ballantyne

    A second collaborative exercise on RNA\\/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5–0.001?l blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA\\/DNA co-extraction or solely RNA extraction

  12. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

  13. Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding: effective tuning of dihedral angle beta in PNAs for DNA/RNA discrimination.

    PubMed

    Govindaraju, T; Madhuri, V; Kumar, Vaijayanti A; Ganesh, Krishna N

    2006-01-01

    [structures: see text] A serious drawback of peptide nucleic acids (PNAs) from an application perspective that has not been adequately dealt with is nondiscrimination of identical DNA and RNA sequences. An analysis of the available X-ray and NMR solution structures of PNA complexes with DNA and RNA suggested that it might be possible to rationally impart DNA/RNA duplex binding selectivity by tuning the dihedral angle beta of the flexible ethylenediamine part of the PNA backbone (II) via suitable chemical modifications. Cyclohexanyl PNAs (chPNAs) with beta approximately = 65 degrees were designed on the basis of this rationale. The chPNAs introduced remarkable differences in duplex stabilities among their DNA and RNA complexes, with melting temperatures (deltaTm(RNA-DNA) = +16-50 degrees C) depending on the number of modifications and the stereochemistry. This is a highly significant, exceptional binding selectivity of a mix sequence of PNA to RNA over the same DNA sequence as that seen to date. In contrast, cyclopentanyl PNAs (cpPNAs) with beta approximately = 25 degrees hybridize to DNA/RNA strongly without discrimination because of the ring puckering of the cyclopentane ring. The high affinity of chPNAs to bind to RNA without losing base specificity will have immediate implications in designing improved PNAs for therapeutic and diagnostic applications. PMID:16388612

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

    PubMed Central

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

    2014-01-01

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

  15. UvrD facilitates DNA repair by pulling RNA polymerase backwards

    PubMed Central

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2015-01-01

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. We further show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes. PMID:24402227

  16. Simultaneous DNA and RNA Mapping of Somatic Mitochondrial Mutations across Diverse Human Cancers

    PubMed Central

    Stewart, James B.; Alaei-Mahabadi, Babak; Sabarinathan, Radhakrishnan; Samuelsson, Tore; Gorodkin, Jan; Gustafsson, Claes M.; Larsson, Erik

    2015-01-01

    Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3? to 5? direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems. PMID:26125550

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

    PubMed

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

    2014-04-01

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

  18. Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

    PubMed

    Radpour, Ramin; Sikora, Michal; Grussenmeyer, Thomas; Kohler, Corina; Barekati, Zeinab; Holzgreve, Wolfgang; Lefkovits, Ivan; Zhong, Xiao Yan

    2009-11-01

    Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome. PMID:19780627

  19. Interaction of antitumor drug Sn(CH 3) 2Cl 2 with DNA and RNA

    NASA Astrophysics Data System (ADS)

    Nafisi, Shohreh; Sobhanmanesh, Amir; Esm-Hosseini, Majid; Alimoghaddam, Kamran; Tajmir-Riahi, Heidar Ali

    2005-08-01

    Sn(CH 3) 2Cl 2 exerts its antitumor activity in a specific way. Unlike anticancer cis-Pt(NH 3) 2Cl 2 drug which binds strongly to the nitrogen atoms of DNA bases, Sn(CH 3) 2Cl 2 shows no major affinity towards base binding. Thus, the mechanism of action by which tinorganometallic compounds exert antitumor activity would be different from that of the cisplatin drug. The aim of this study was to examine the binding of Sn(CH 3) 2Cl 2 with calf thymus DNA and yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentrations of DNA and RNA and various molar ratios of Sn(CH 3) 2Cl 2/DNA (phosphate) and Sn(CH 3) 2Cl 2/RNA of 1/40, 1/20, 1/10, 1/5. Fourier transform infrared (FTIR) and UV-visible difference spectroscopic methods were used to determine the Sn(CH 3) 2Cl 2 binding mode, binding constant, sequence selectivity and structural variations of Sn(CH 3) 2Cl 2/DNA and Sn(CH 3) 2Cl 2/RNA complexes in aqueous solution. Sn(CH 3) 2Cl 2 hydrolyzes in water to give Sn(CH 3) 2(OH) 2 and [Sn(CH 3) 2(OH)(H 2O) n] + species. Spectroscopic evidence showed that interaction occurred mainly through (CH 3) 2Sn(IV) hydroxide and polynucleotide backbone phosphate group with overall binding constant of K(Sn(CH 3) 2Cl 2-DNA)=1.47×10 5 M -1 and K(Sn(CH 3) 2Cl 2-RNA)=7.33×10 5 M -1. Sn(CH 3) 2Cl 2 induced no biopolymer conformational changes with DNA remaining in the B-family structure and RNA in A-conformation upon drug complexation.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    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

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

  2. Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.

    PubMed

    Corrick, C M; Silvestro, M J; Lahoud, M H; Allen, G J; Tymms, M J; Kola, I

    1996-01-01

    Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones. PMID:8720109

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1985-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    PubMed Central

    Munafó, Daniela B.; Robb, G. Brett

    2010-01-01

    Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2?-O-methylation of the 3?-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2?-O-methyl modification of small RNA 3?-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples. PMID:20921270

  7. Mapping of promoter sites utilized by T3 RNA polymerase on T3 DNA.

    PubMed Central

    Bailey, J N; McAllister, W T

    1980-01-01

    Promoter locations for the T3 RNA polymerase on the physical map of T3 DNa have been determined. Through the use of conditions favoring the synthesis of RNA from the class II region, an agarose-formaldehyde gel system which improves the resolution of high molecular weight RNAs, and template DNA that was cut by one of several restriction endonucleases prior to transcription, seventeen promoter locations for the T3 RNA polymerase have been mapped. Ten promoters have been identified in the class II region and one promotor has been identified in the class II region and one promotor has been identified in the early (class I) region. The locations of previously mapped class III promoters and the internal termination signal for the T3 RNA polymerase have been mapped more precisely than in previous reports. The resulting transcription map demonstrates a striking similarity to the transcription map of bacteriophage T7. Images PMID:7443532

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

    PubMed Central

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

    2010-01-01

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

  9. In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.

    PubMed

    Anders, Carolin; Niewoehner, Ole; Jinek, Martin

    2015-01-01

    The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes. PMID:26068752

  10. Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

    PubMed Central

    Goldman, W E; Goldberg, G; Bowman, L H; Steinmetz, D; Schlessinger, D

    1983-01-01

    Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats. PMID:6621535

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

    Microsoft Academic Search

    Ming Li; Baerbel Rohrer

    2006-01-01

    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

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

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

    2013-01-01

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

  14. Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

    PubMed Central

    James, T C; Bond, U M; Maack, C A; Applebaum, S W; Tata, J R

    1982-01-01

    Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin. Images Fig. 1. Fig. 2. Fig. 3. PMID:6184048

  15. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices. PMID:16269194

  16. The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions

    PubMed Central

    Kljashtorny, Vladislav; Nikonov, Stanislav; Ovchinnikov, Lev; Lyabin, Dmitry; Vodovar, Nicolas; Curmi, Patrick; Manivet, Philippe

    2015-01-01

    The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids. PMID:26147853

  17. Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry.

    PubMed

    Nowak, El?bieta; Miller, Jennifer T; Bona, Marion K; Studnicka, Justyna; Szczepanowski, Roman H; Jurkowski, Jakub; Le Grice, Stuart F J; Nowotny, Marcin

    2014-04-01

    Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)-containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract-containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer. PMID:24608367

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

    SciTech Connect

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

    1994-12-31

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

  19. Stabilizing RNA by the sonochemical formation of RNA nanospheres.

    PubMed

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

    2011-04-18

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

  20. Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity

    PubMed Central

    McDougall, William M.; Smith, Harold C.

    2011-01-01

    APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cytoplasmic P-bodies and stress granules. Using a defined in vitro system, we show that RNA alone was sufficient to suppress A3G deaminase activity and did so in an RNA concentration-dependent manner. RNAs of diverse sequences and as short as 25 nucleotides were effective inhibitors. Native PAGE analyses showed that RNA formed ribonucleoprotein complexes with A3G and in so doing prevented ssDNA substrates from binding to A3G. The data provided direct evidence that A3G binding to cellular RNAs constituted a substantial impediment to the enzyme’s ability to interact with ssDNA. PMID:21856286

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

    Microsoft Academic Search

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

    1990-01-01

    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

  2. Differentiation of strains of tick-borne encephalitis virus by means of RNA-DNA hybridization

    Microsoft Academic Search

    V. A. Shamanin; A. G. Pletnev; S. G. Rubin; V. I. Zlobin

    1990-01-01

    Cloned cDNA and synthetic deoxyoligonucleotides, complementary to various parts of the genomic RNA of tick-borne encephalitis virus (TBEV), strain Sol)in, were used to distinguish between strains of TBEV and other flaviviruses. The cDNA probe hybridized with strains of TBEV and related flaviviruses of the TBE complex except for Powassan virus, and it did not react with flaviviruses of the Japanese

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

    PubMed Central

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

    2012-01-01

    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. PMID:22885844

  4. Histidine-rich cationic amphipathic peptides for plasmid DNA and siRNA delivery.

    PubMed

    Kichler, Antoine; Mason, A James; Marquette, Arnaud; Bechinger, Burkhard

    2013-01-01

    Amphipathic, pH-responsive, membrane-active peptides such as LAH4 and derivatives thereof have the ability to effectively deliver genes and small interfering RNA (siRNA) into mammalian cells. Their ability to bind and protect nucleic acids and then disrupt membranes when activated at low pH enables them to harness the endocytic machinery to deliver cargo efficiently and with low associated toxicity. This chapter describes protocols for the chemical synthesis of transfection peptides of the LAH4 family, complex formation with nucleic acids, and their use for the in vitro delivery of either plasmid DNA or siRNA into mammalian cell lines. PMID:23070765

  5. RNA decay mechanisms: Specicity through diversity Nature has invented the polymerization of RNA from a DNA

    E-print Network

    Mühlemann, Oliver

    Editorial RNA decay mechanisms: Specicity through diversity Nature has invented the polymerization, and ­ we tend to forget ­ as enzymes that help us to digest our daily bread. The purpose of this special

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

    E-print Network

    Stroud, Todd

    2010-01-20

    of the target mRNA through the ribosome, therefore preventing translation of the mRNA into protein. Unlike siRNA that generally result in degradation, miRNA simply prevent translation by the ribosome (Hannon, Rivas et al. 2006; Scherr and Eder 2007... using a computer program on the Cold Spring Harbor website (http://www.cshl.edu/public/SCIENCE/hannon.html). A previous study in our lab suggested that two of the shRNA sequences effectively knocked down the expression of DNMT1 protein. These two sh...

  7. A Course on Macromolecules.

    ERIC Educational Resources Information Center

    Horta, Arturo

    1985-01-01

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

  8. RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

    PubMed

    Haas, C; Hanson, E; Anjos, M J; Bär, W; Banemann, R; Berti, A; Borges, E; Bouakaze, C; Carracedo, A; Carvalho, M; Castella, V; Choma, A; De Cock, G; Dötsch, M; Hoff-Olsen, P; Johansen, P; Kohlmeier, F; Lindenbergh, P A; Ludes, B; Maroñas, O; Moore, D; Morerod, M-L; Morling, N; Niederstätter, H; Noel, F; Parson, W; Patel, G; Popielarz, C; Salata, E; Schneider, P M; Sijen, T; Sviežena, B; Turanská, M; Zatkalíková, L; Ballantyne, J

    2012-01-01

    A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 ?l blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology. PMID:21459062

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

    Microsoft Academic Search

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

    1997-01-01

    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

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

    E-print Network

    Weiblen, George D

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

  11. DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips.

    PubMed Central

    Tanimoto, E. Y.; Rost, T. L.; Comai, L.

    1993-01-01

    Histone H2A mRNA is selectively expressed in scattered subpopulations of cells in the pea (Pisum sativum) root apical meristem. To study whether this specific expression was associated with the cell cycle, a double-labeling technique was used to identify cells replicating DNA during S phase and those expressing H2A mRNA. Cells in S phase were detected by [3H]thymidine incorporation and autoradiography, whereas cells containing H2A mRNA were identified by in situ hybridization using digoxigenin-labeled probes. Approximately 92% of the [3H]thymidine-labeled S-phase cells expressed H2A mRNA and 85% of cells that expressed H2A mRNA were in S phase. In root tissue located basal to the promeristem, synchronous co-located expression was observed in scattered packets of proliferating cells. Furthermore, neither H2A mRNA nor S-phase cells could be detected within the quiescent center or mature root cap. When DNA synthesis was inhibited with hydroxyurea, a commensurate and specific decrease in steady-state levels of H2A mRNA was found. We conclude that cell-specific expression of pea histone H2A mRNA is replication dependent and that H2A mRNA is transiently accumulated during a period of the cell cycle that mostly overlaps the S phase. We propose that the overlap between H2A expression and S phase could occur if H2A mRNA accumulation began in late G1 and abated in late S. PMID:12232021

  12. Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase

    PubMed Central

    Mallam, Anna L; Sidote, David J; Lambowitz, Alan M

    2014-01-01

    How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the ‘closed-state’ helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions. DOI: http://dx.doi.org/10.7554/eLife.04630.001 PMID:25497230

  13. Widespread RNA and DNA Sequence Differences in the

    E-print Network

    Zhang, Jianzhi

    and in different cell types, including primary skin cells and brain tissues. Using mass spectrometry, we detected of these were seen in B cells, as well as in primary skin cells and brain tissues from a separate set of individuals and in expressed se- quence tags (ESTs) from cDNA libraries of var- ious cell types. About 43

  14. Genome Analysis Transfer RNA gene recruitment in mitochondrial DNA

    E-print Network

    Lavrov, Dennis V.

    N K atp8 atp6 cox3 Q W cob T atp9 nad4nad6 nad3 nad4L cox1 D nad1 Y M nad2 nad5 rns L2F GV R2 S1 L1 alanine tRNAs. Protein (blue) and rRNA (green) genes are abbreviated as follows: atp6, 8­9 (genes encoding

  15. Thermodynamics of RNA\\/DNA hybridization in high-density oligonucleotide microarrays

    Microsoft Academic Search

    Enrico Carlon; Thomas Heim

    2006-01-01

    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

  16. Isolation of DNA-dependent RNA polymerase from the thermophilic actinomycete Thermomonospora curvata

    Microsoft Academic Search

    M. Krausová; M. Klégr; J. Spížek

    1994-01-01

    DNA-Dependent RNA polymerase (EC 2.7.7.6) was isolated fromThermomonospora curvata. The purification steps included precipitation with Polymin P, elution of the precipitate with 0.3 mol\\/L KCl, precipitation\\u000a with ammonium sulfate, affinity chromatography on heparin-agarose and molecular filtration on Biogel A 1.5m.

  17. DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

    E-print Network

    DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA Yaqing Du1¤a, United States of America Abstract Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes

  18. The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site

    PubMed Central

    Tupin, Audrey; Gualtieri, Maxime; Leonetti, Jean-Paul; Brodolin, Konstantin

    2010-01-01

    Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the ?70 subunit region 3.2 and the RNAP ?? subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the ‘closed'-promoter complex to the transcriptionally competent ‘open' complex and blocks ?70-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the ?? subunit switch-2 element and the ?70 subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process. PMID:20562828

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

    Microsoft Academic Search

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

    1996-01-01

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

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

    SciTech Connect

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

    2012-12-01

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

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

    PubMed

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

    2014-08-01

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

  2. Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode.

    PubMed

    Miao, Peng; Wang, Bidou; Meng, Fanyu; Yin, Jian; Tang, Yuguo

    2015-03-18

    MicroRNAs are a class of evolutionally conserved, small noncoding RNAs involved in the regulation of gene expression and affect a variety of biological processes including cellular differentiation, immunological response, tumor development, and so on. Recently, microRNAs have been identified as promising disease biomarkers. In this work, we have fabricated a novel electrochemical method for ultrasensitive detection of microRNA. Generally, a DNA tetrahedron decorated gold electrode is employed as the recognition interface. Then, hybridizations between DNA tetrahedron, microRNA, and primer probe initiate rolling circle amplification (RCA) on the electrode surface. Silver nanoparticles attached to the RCA products provide significant electrochemical signals and a limit of detection as low as 50 aM is achieved. Moreover, homology microRNA family members with only one or two mismatches can be successfully distinguished. Therefore, this proposed method reveals great advancements toward improved disease diagnosis and prognosis. PMID:25692917

  3. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

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

    2007-04-06

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

  4. Comparative Dynamics and Sequence Dependence of DNA and RNA Binding to Single Walled Carbon Nanotubes

    PubMed Central

    Landry, Markita P.; Vukovi?, Lela; Kruss, Sebastian; Bisker, Gili; Landry, Alexandra M.; Islam, Shahrin; Jain, Rishabh; Schulten, Klaus; Strano, Michael S.

    2015-01-01

    Noncovalent polymer-single walled carbon nanotube (SWCNT) conjugates have gained recent interest due to their prevalent use as electrochemical and optical sensors, SWCNT-based therapeutics, and for SWCNT separation. However, little is known about the effects of polymer-SWCNT molecular interactions on functional properties of these conjugates. In this work, we show that SWCNT complexed with related polynucleotide polymers (DNA, RNA) have dramatically different fluorescence stability. Surprisingly, we find a difference of nearly 2500-fold in fluorescence emission between the most fluorescently stable DNA-SWCNT complex, C30 DNA-SWCNT, compared to the least fluorescently stable complex, (AT)7A-(GU)7G DNA-RNA hybrid-SWCNT. We further reveal the existence of three regimes in which SWCNT fluorescence varies nonmonotonically with SWCNT concentration. We utilize molecular dynamics simulations to elucidate the conformation and atomic details of SWCNT-corona phase interactions. Our results show that variations in polynucleotide sequence or sugar backbone can lead to large changes in the conformational stability of the polymer SWCNT corona and the SWCNT optical response. Finally, we demonstrate the effect of the coronae on the response of a recently developed dopamine nanosensor, based on (GT)15 DNA- and (GU)15 RNA-SWCNT complexes. Our results clarify several features of the sequence dependence of corona phases produced by polynucleotides adsorbed to single walled carbon nanotubes, and the implications for molecular recognition in such phases. PMID:26005509

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

    PubMed

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

    2013-06-11

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    1981-06-01

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

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

    PubMed Central

    Figiel, Ma?gorzata; Nowotny, Marcin

    2014-01-01

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

  9. A subset of conserved tRNA genes in plastid DNA of nongreen plants.

    PubMed Central

    Lohan, A J; Wolfe, K H

    1998-01-01

    The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion. PMID:9725858

  10. Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens.

    PubMed

    Satra, Maria; Vamvakopoulou, Dimitra N; Sioutopoulou, Despina O; Kollia, Panagoula; Kiritsaka, Aspasia; Sotiriou, Sotirios; Antonakopoulos, Georgios; Alexandris, Elias; Costantoulakis, Pantelis; Vamvakopoulos, Nicholas C

    2009-01-01

    We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence. PMID:19762162

  11. Template specificities of a RNA-directed DNA polymerase from a human homologous mixed mesodermal sarcoma.

    PubMed

    Weiss, G B; Carr, B K; Hannigan, E V

    1983-01-01

    A RNA-directed DNA polymerase was partially purified from a human homologous, mixed mesodermal sarcoma by DEAE-cellulose chromatography after sucrose density centrifugation. The enzyme transcribed poly(rA) most effectively but also transcribed poly(rI), poly(dA) and poly(rG) and to a lesser extent, poly(rmC). It was unable to transcribe poly(rU). The product with poly(rA) as template contained large material (greater than 28S) in addition to some proper size product demonstrating a slippage reaction. This pattern of transcription, while similar to avian myeloblastosis virus DNA polymerase, reveals qualitative differences making direct extrapolation from studies with animal oncornaviruses to human cancer difficult. In this paper, the detection and purification of RNA-directed DNA polymerase from a patient with an uncommon uterine sarcoma is reported along with the template specificities of the enzyme. PMID:6191264

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

    PubMed Central

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

    2014-01-01

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

  13. Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs.

    PubMed

    Strom, Shane; Shiskova, Evgenia; Hahm, Yaeeun; Grover, Neena

    2015-07-01

    Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl2 or 1 M KCl. The [Formula: see text] loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ??G° values relative to 1 M KCl were 0.47-2.06 kcal/mol more favorable for the RNA bulge loops. The [Formula: see text] loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride. PMID:26022248

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

    PubMed Central

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

    2013-01-01

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

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

    E-print Network

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

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

    Microsoft Academic Search

    Sripriya Venkiteswaran; Thresia Thomas; T. J. Thomas

    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

  17. Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis.

    PubMed

    Qiao, Man; Luo, Dan; Kuang, Yi; Feng, Haiyan; Luo, Guangping; Liang, Peng

    2015-06-18

    p53 tumor-suppressor gene is a master transcription factor which controls cell cycle progression and apoptosis. killin was discovered as one of the p53 target genes implicated in S-phase control coupled to cell death. Due to its extreme proximity to pten tumor-suppressor gene on human chromosome 10, changes in epigenetic modification of killin have also been linked to Cowden syndrome as well as other human cancers. Previous studies revealed that Killin is a high-affinity DNA-binding protein with preference to single-stranded DNA, and it inhibits DNA synthesis in vitro and in vivo. Here, co-localization studies of RFP-Killin with either GFP-PCNA or endogenous single-stranded DNA binding protein RPA during S-phase show that Killin always adopts a mutually exclusive punctuated nuclear expression pattern with the 2 accessory proteins in DNA replication. In contrast, when cells are not in S-phase, RFP-Killin largely congregates in the nucleolus where rRNA transcription normally occurs. Both of these cell cycle specific localization patterns of RFP-Killin are stable under high salt condition, consistent with Killin being tightly associated with nucleic acids within cell nuclei. Together, these cell biological results provide a molecular basis for Killin in competitively inhibiting the formation of DNA replication forks during S-phase, as well as potentially negatively regulate RNA synthesis during other cell cycle phases. PMID:25945611

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

    Microsoft Academic Search

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

    2009-01-01

    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

  19. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation.

    PubMed

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

    2014-03-01

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

  20. Nucleocytoplasmic transport of macromolecules.

    PubMed Central

    Corbett, A H; Silver, P A

    1997-01-01

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

  1. The differential processing of telomeres in response to increased telomeric transcription and RNA–DNA hybrid accumulation

    PubMed Central

    Balk, Bettina; Dees, Martina; Bender, Katharina; Luke, Brian

    2014-01-01

    Telomeres are protective nucleoprotein structures at the ends of eukaryotic chromosomes. Despite the heterochromatic state of telomeres they are transcribed, generating non-coding telomeric repeat-containing RNA (TERRA). Strongly induced TERRA transcription has been shown to cause telomere shortening and accelerated senescence in the absence of both telomerase and homology-directed repair (HDR). Moreover, it has recently been demonstrated that TERRA forms RNA–DNA hybrids at chromosome ends. The accumulation of RNA–DNA hybrids at telomeres also leads to rapid senescence and telomere loss in the absence of telomerase and HDR. Conversely, in the presence of HDR, telomeric RNA–DNA hybrid accumulation and increased telomere transcription promote telomere recombination, and hence, delayed senescence. Here, we demonstrate that despite these similar phenotypic outcomes, telomeres that are highly transcribed are not processed in the same manner as those that accumulate RNA–DNA hybrids. PMID:24525824

  2. RNA involvement in T4 DNA synthesis in toluene-treated cells.

    PubMed

    Dicou, E

    1980-01-01

    In T4-infected cells made permeable with toluene, pulses with [(alpha-32P deoxyribonucleoside triphosphates demonstrated covalent linkage of RNA to DNA of the Okazaki fragments. Analysis of the transfer of the 32P label to the 2'(3') ribonucleoside monophosphates indicated that the 3'-end of the RNA primer is heterogeneous. The most frequently encountered ribonucleotide was rCMP, but also transfer to rUMP, rAMP and rGMP occurred at different frequencies. In contrast, no heterogeneity was observed for the deoxyribonucleoside at the RNA-DNA junction. Of all the [to-32P] deoxyribonucleoside triphosphates tested, transfer of the 32P label to 2'(3') rNMPs was predominant when [alpha32P] dGTP was the substrate, indicating that the deoxyribonucleoside most frequently encountered at the RNA-DNA linkage is dG. These observations suggest that the starts for the Okazaki fragments may occur at unique sites of the T4 genome. PMID:17941178

  3. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

    PubMed

    He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

    2015-08-01

    Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery. PMID:25937003

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

    PubMed Central

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

    1986-01-01

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

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

    SciTech Connect

    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

    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.

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

    PubMed Central

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

    2012-01-01

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

  7. Wrapping of Promoter DNA around the RNA Polymerase II Initiation Complex Induced by TFIIF

    PubMed Central

    Robert, François; Douziech, Maxime; Forget, Diane; Egly, Jean-Marc; Greenblatt, Jack; Burton, Zachary F.; Coulombe, Benoit

    2015-01-01

    Summary The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein–DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions ?40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation. PMID:9774972

  8. DNA-Dependent RNA Polymerase Detects Hidden Giant Viruses in Published Databanks

    PubMed Central

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

    2014-01-01

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

  9. MicroRNA-376a Sensitizes Cells Following DNA Damage by Downregulating MEPE Expression

    PubMed Central

    Sheng, Jipo; Luo, Wei; Yu, Fang; Gao, Ning

    2013-01-01

    Abstract MicroRNAs (miRNAs) are a class of endogenous molecules that post-transcriptionally regulate target gene expression and play an important role in many developmental processes. Matrix extracellular phosphoglycoprotein (MEPE) is related to bone metabolism. We recently reported that MEPE protects cells from DNA damage-induced killing. The purpose of this study is to investigate whether miRNAs targeting MEPE play an important role in DNA damage response. We report in this study that miR-376a directly targets MEPE, and overexpression of miR-376a reduces the G2 arrest of the cells and sensitizes the cells to DNA damage-induced killing. These results indicate an association of MEPE gene inactivation with decreased survival after DNA damage and also provide useful information for miRNA-based drug development: a new target for sensitizing human tumor cells to radiotherapy or chemotherapy. PMID:23570370

  10. 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

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

    2008-12-12

    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

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

    PubMed Central

    2013-01-01

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

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

    SciTech Connect

    Smith, O.P.

    1987-01-01

    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.

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

    PubMed Central

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

    2005-01-01

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

  14. Structural and functional analyses of the interaction of archaeal RNA polymerase with DNA

    PubMed Central

    Wojtas, Magdalena N.; Mogni, Maria; Millet, Oscar; Bell, Stephen D.; Abrescia, Nicola G. A.

    2012-01-01

    Multi-subunit RNA polymerases (RNAPs) in all three domains of life share a common ancestry. The composition of the archaeal RNAP (aRNAP) is not identical between phyla and species, with subunits Rpo8 and Rpo13 found in restricted subsets of archaea. While Rpo8 has an ortholog, Rpb8, in the nuclear eukaryal RNAPs, Rpo13 lacks clear eukaryal orthologs. Here, we report crystal structures of the DNA-bound and free form of the aRNAP from Sulfolobus shibatae. Together with biochemical and biophysical analyses, these data show that Rpo13 C-terminus binds non-specifically to double-stranded DNA. These interactions map on our RNAP–DNA binary complex on the downstream DNA at the far end of the DNA entry channel. Our findings thus support Rpo13 as a RNAP–DNA stabilization factor, a role reminiscent of eukaryotic general transcriptional factors. The data further yield insight into the mechanisms and evolution of RNAP–DNA interaction. PMID:22848102

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

    PubMed Central

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

    2013-01-01

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

  16. Bioactivation of 2-nitrofluorene to reactive intermediates that bind covalently to DNA, RNA and protein in vitro and in vivo in the rat.

    PubMed

    Wierckx, F C; Wedzinga, R; Meerman, J H; Mulder, G J

    1990-01-01

    The activation of 2-nitrofluorene (2-NF) to reactive intermediates that bind covalently to DNA, RNA and protein has been investigated both in vitro and in the rat in vivo. In vitro, such binding was catalyzed by the hepatic microsomal fraction, was NADPH dependent and could be inhibited by SKF 525A, an inhibitor of cytochrome P450. The generation of reactive intermediates therefore is most likely catalyzed by cytochrome P450. Covalent binding of 2-NF could not be prevented by glutathione, N-acetylcysteine and other thiol-containing compounds. It could be partially prevented by guanosine, presumably because it traps the reactive intermediate(s). Under normal oxygenation conditions 2-NF was also covalently bound in freshly isolated hepatocytes; pretreatment of rats with an inducer of cytochrome P450, Aroclor 1254, gave rise to a higher rate of covalent binding in hepatocytes. Covalent binding of 2-NF to cellular macromolecules also occurred in vivo, both after oral and i.v. administration. Pentachlorophenol, a selective sulfation inhibitor, did not influence the covalent binding of 2-NF; therefore, the reactive intermediate is not formed by sulfation of N-hydroxy-2-acetylaminofluorene, which could be a metabolite of 2-NF. It is concluded that the reactive intermediates most likely can be formed from 2-NF by the cytochrome P450 enzyme system. PMID:1688520

  17. Differentiating the Protein Coding and Noncoding RNA Segments of DNA Using Shannon Entropy

    NASA Astrophysics Data System (ADS)

    Mazaheri, P.; Shirazi, A. H.; Saeedi, N.; Reza Jafari, G.; Sahimi, Muhammad

    The complexity of DNA sequences is evaluated in order to differentiate between protein-coding and noncoding RNA segments. The method is based on computing the Shannon entropy of the sequences. By comparing the entropy of the original sequence with that of its shuffled one, we identify the source of the difference between the two segments and their relative contributions to the sequence. To demonstrate the method, the DNA sequences of the bacterium Clostridium difficile 630 (G + C = 29.1%) and Bdellovibrio bacteriovorus (G + C = 50.6%) are analyzed, which are representatives of bacteria with unbalanced and balanced nucleotide content, respectively. It is shown that in both bacteria, regardless of nucleotide content, ?rS — the relative difference of the two entropies — is significantly greater in protein-coding regions, when compared with noncoding RNA segments.

  18. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    PubMed

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells. PMID:25764216

  19. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    PubMed

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-27

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells. PMID:24531370

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

    E-print Network

    Stroud, Todd

    2010-01-20

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

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

    Microsoft Academic Search

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

    2012-01-01

    An RNA–DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the

  2. Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens

    Microsoft Academic Search

    Maria Satra; Dimitra N. Vamvakopoulou; Despina O. Sioutopoulou; Panagoula Kollia; Aspasia Kiritsaka; Sotirios Sotiriou; Georgios Antonakopoulos; Elias Alexandris; Pantelis Costantoulakis; Nicholas C. Vamvakopoulos

    2009-01-01

    We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin

  3. A Relationship Between DNA Helix Stability and Recognition Sites for RNA Polymerase

    Microsoft Academic Search

    H. J. Vollenweider; M. Fiandt; W. Szybalski

    1979-01-01

    The RNA polymerase binding sites on the DNA of (i) the aroE-trkA-spc segment of the Escherichia coli genome, (ii) transposon Tn3, (iii) plasmid ColEl, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the BAC technique; these maps were compared with the maps of the early-melting regions for the same genomes. The results indicate that in

  4. DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

    Microsoft Academic Search

    Luis Oñate-Sánchez; Jesús Vicente-Carbajosa

    2008-01-01

    BACKGROUND: High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material

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

    PubMed Central

    Chícharo, Maria Alexandra; Chícharo, Luis

    2008-01-01

    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

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

    PubMed

    Chícharo, Maria A; Chícharo, Luis

    2008-08-01

    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

  7. RNA/DNA ratio as biomarkers for periphyton and macroinvertebrate growth

    NASA Astrophysics Data System (ADS)

    Mewes, Daniela; Winkelmann, Carola

    2015-04-01

    A biocenosis is a complex assembly of organisms driven and shaped by numerous processes and interactions. Yet, in order to describe the biocenosis of a stream often only state variables, such as algal biomass or invertebrate diversity and abundance, are measured. But these variables fail to provide much needed information on those driving processes. Because processes such as growth of periphyton and invertebrates can hardly be measured directly in the field, the use of biomarkers is a promising approach to quantify biological rates under natural conditions. Periphyton represents the main food source for invertebrate grazers and periphyton growth rate rather than standing stocks alone allows the estimation of the availability of this resource. A linear relationship of RNA/DNA ratios and growth rate has previously been established for single species cultures of algae and bacteria but not for naturally occurring freshwater periphyton assemblages. In this study it could be shown that linear relationships of RNA/DNA ratios and growth rate are also valid for naturally occurring freshwater periphyton assemblages and can be used as biomarkers for periphyton growth rate. Moreover, recent results indicate that the RNA/DNA ratio might also be used as biomarker for invertebrates, because high-quality food was observed to increase the RNA/DNA ratios of the freshwater amphipod Dikerogammarus villosus. These are very promising results with regard to the usefulness and applicability of biomarkers ecosystem analysis in running waters. Additional biomarkers allowing the analysis of further processes and interactions within the food web such as PLFAs (phospholipid-fatty acids), neutral lipids and PUFAs (polyunsaturated fatty acids) are to be tested for their applicability in stream ecosystems.

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

    PubMed Central

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

    2014-01-01

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

  9. Nucleic Acid Sequence-Based Amplification Assay for Human Papillomavirus mRNA Detection and Typing: Evidence for DNA Amplification?

    PubMed Central

    Boulet, Gaëlle A. V.; Micalessi, Isabel M.; Horvath, Caroline A. J.; Benoy, Ina H.; Depuydt, Christophe E.; Bogers, Johannes J.

    2010-01-01

    Human papillomavirus (HPV) E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA. This study evaluated the RNA specificity of nucleic acid sequence-based amplification (NASBA)-based HPV detection using HPV DNA plasmids (HPV type 16 [HPV16], HPV18, HPV31, HPV33, and HPV45) and nucleic acid extracts of several cell lines, which were systematically subjected to enzymatic treatments with DNase and RNase. HPV plasmid dilutions (106 to 100 copies/?l) and nucleic acid extracts (total DNA, RNA-free DNA, total RNA, and DNA-free RNA) of unfixed and fixed (PreServCyt and SurePath) HaCaT, HeLa, and CaSki cells were tested with the NucliSENS EasyQ HPV test. The RNA-free DNA extracts of HeLa and CaSki cells could be amplified by HPV18 and -16 NASBA, respectively. Fixation of the cells did not influence NASBA. All HPV plasmids could be detected with NASBA. Based on the plasmid dilution series, a lower detection limit of 5 × 103 HPV DNA copies could be determined. Our study identified viral double-stranded DNA as a possible target for NASBA-based HPV detection. The differences in diagnostic accuracy between the NASBA-based tests and conventional HPV DNA detection assays seem to be attributable not to the more specific amplification of viral mRNA but to the limited type range and the lower analytical sensitivity for HPV DNA. PMID:20463156

  10. DNA sequence of the tandem ribosomal RNA promoter for B. subtilis operon rrnB.

    PubMed Central

    Stewart, G C; Bott, K F

    1983-01-01

    A new ribosomal RNA operon designated rrnB has been identified by screening a Charon 4a library of cloned B. subtilis sequences. Clones containing the promoter region of this operon are unstable in E. coli unless a special vector possessing a transcriptional terminator is used. DNA sequence data suggests that this operon contains two tandem putative promotor regions not unlike those found in E. coli. There are 92 base pairs separating the two "-10 regions" of the promotors. The second is 180 bp upstream from the start site for mature 16S RNA. A potential 29 base pair stem structure necessary for processing of the mature 16S RNA sequence can also be predicted from this analysis. Images PMID:6312417

  11. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    PubMed

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. PMID:23036990

  12. Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa.

    PubMed

    Rollins, MaryClare F; Schuman, Jason T; Paulus, Kirra; Bukhari, Habib S T; Wiedenheft, Blake

    2015-02-27

    The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide. PMID:25662606

  13. Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa

    PubMed Central

    Rollins, MaryClare F.; Schuman, Jason T.; Paulus, Kirra; Bukhari, Habib S.T.; Wiedenheft, Blake

    2015-01-01

    The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide. PMID:25662606

  14. Microbial rRNA:rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils.

    PubMed

    Dlott, Glade; Maul, Jude E; Buyer, Jeffrey; Yarwood, Stephanie

    2015-08-01

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communities. 'Activity ratios' were calculated for bacteria and archaea in soil sampled from a tropical rainforest and temperate agricultural field and incubated for one year at two levels of moisture availability and with and without carbon additions. Prior to calculating activity ratios, we corrected the relative abundances of OTUs to account for multiple copies of the 16S gene per genome. Although necessary to ensure accurate activity ratios, this correction did not change our interpretation of differences in microbial community composition across treatments. Activity ratios in this study were lower than those previously published (0.0003-210, logarithmic mean=0.24), suggesting significant extracellular DNA preservation. After controlling for the influence of individual incubation jars, significant differences in activity ratios between all members of each phylum were observed. Planctomycetes and Firmicutes had the highest activity ratios and Crenarchaeota had the lowest activity overall. Our results suggest that greater caution should be taken in interpreting soil microbial community data derived from extracted DNA. Indirect extraction methods may be useful in ensuring that microbes identified from extracellular DNA are not erroneously interpreted as components of an active microbial community. PMID:26055315

  15. Single-Stranded DNA and RNA Targeted Triplex-Formation: UV, CD and Molecular Modeling Studies of Foldback Triplexes Containing Different RNA, 2?-OMe-RNA and DNA Strand Combinations

    Microsoft Academic Search

    Ekambar R. Kandimalla; Ganesh Venkataraman; V. Sasisekharan; Sudhir Agrawal

    1997-01-01

    We studied the influence of different 2?-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D D:D, R* D:D, D D:R*, R* D:R*, D R:D, R* R:D, DR:R*, and R*-R:R*; where the first, middle, and

  16. Brain Single Photon Emission Computed Tomography in Patients with A3243G Mutation in Mitochondrial DNA tRNA

    Microsoft Academic Search

    Peterus Thajeb; Ming-Che Wu; Bing-Fu Shih; Chin-Yuan Tzen; Ming-Fu Chiang; Rey-Yue Yuan

    2005-01-01

    Brain single photon emission computed tomography (SPECT) studies were conducted in three patients with A3243G mutation of the mitochondrial (mt) DNA tRNA. All were born to mothers suffering from chronic progressive external ophthalmoplegia (CPEO) with the same A3243G point mutation of the mtDNA tRNA. The first case manifested clinically with MELAS, the second case manifested with CPEO, and third case

  17. Selective Nucleic Acid Removal via Exclusion (SNARE): Capturing mRNA and DNA from a single sample

    PubMed Central

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

    2013-01-01

    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 non-dilutive re-interrogation 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, 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

  18. D-RNAi (Messenger RNA-antisense DNA Interference) as a Novel Defense System Against Cancer and Viral Infections

    Microsoft Academic Search

    Shi-Lung Lin; Shao-Yao Ying; Cheng-Ming Chuong

    2001-01-01

    D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4(+) T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was

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

    Microsoft Academic Search

    M. J. Dagg; J. L. Littlepage

    1972-01-01

    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

  20. ACCOUNT AND PERSPECTIVE Macromolecule Mass Spectrometry

    E-print Network

    Karypis, George

    ACCOUNT AND PERSPECTIVE Macromolecule Mass Spectrometry: Citation Mining of User Documents Ronald N the identification and structural analysis of biological macromolecules. In particular, Fenn and Tanaka focused

  1. Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

    PubMed

    Zelenka, Jaroslav; Alán, Lukáš; Jab?rek, Martin; Ježek, Petr

    2014-04-01

    Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import. PMID:24562889

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

    PubMed

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

    2014-05-01

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

  3. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    SciTech Connect

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-03-24

    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.

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed

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

    2012-04-01

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

  6. RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

    PubMed

    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; Maroñas, 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

    2013-02-01

    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

  7. In vitro tRNA methylation assay with the Entamoeba histolytica DNA and tRNA methyltransferase Dnmt2 (Ehmeth) enzyme.

    PubMed

    Tovy, Ayala; Hofmann, Benjamin; Helm, Mark; Ankri, Serge

    2010-01-01

    Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, Dictyostelium discoideum and Drosophila. Our recent work has shown that Ehmeth methylates tRNA(Asp), and this finding indicates that this enzyme has a dual DNA/tRNA(Asp) methyltransferase activity. This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity. PMID:21048666

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

    E-print Network

    Lowenberger, Carl

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

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

    PubMed

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

    2015-02-28

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

  10. Monomers in coal macromolecules

    SciTech Connect

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

    1991-01-01

    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 combustion. In addition to characterization of the pyrolysis fragments, the solvent extractable material which may or may not be thermally extractable requires detailed characterization. The approaches used in this study rely on a number of mass spectrometric techniques, including: fast atom bombardment (FAB) MS, laser desorption (LD) time-of-flight MS, and desorption chemical ionization (DCI) MS, along with tandem MS and high resolution (HR) MS. The eight coals from the Argonne Premium Coal Sample Bank are used in this study along with these coals demineralized, and solvent extracted and the extracts themselves. 10 refs., 2 figs., 1 tab.

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

    SciTech Connect

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

    2010-03-08

    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.

  12. Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems.

    PubMed

    Leung, Alex K K; Tam, Yuen Yi C; Chen, Sam; Hafez, Ismail M; Cullis, Pieter R

    2015-07-16

    Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP-siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP-siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP-siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a "currant bun" morphology as visualized by cryo-TEM. This structure is fully consistent with LNP-siRNA structure predicted by molecular modeling. PMID:26087393

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

    SciTech Connect

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

    1991-01-01

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

  14. Mechanism and function of oxidative reversal of DNA and RNA methylation.

    PubMed

    Shen, Li; Song, Chun-Xiao; He, Chuan; Zhang, Yi

    2014-01-01

    The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/?-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation. PMID:24905787

  15. Sequence-independent upstream DNA-CTD interactions strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association

    Microsoft Academic Search

    Wilma Ross; Richard L. Gourse

    2005-01-01

    The C-terminal domains of the two -subunits (CTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA-CTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA-CTD interactions to promoter

  16. Structure and biology of cartilage and bone matrix noncollagenous macromolecules

    Microsoft Academic Search

    DICK HEINEGARD; AKE OLDBERG

    Over recent years a number of cartilage and bone matrix molecules have been identified and characterized. These include major constituents such as collagens and pro- teoglycans as well as a number of less-abundant matrix proteins. In several cases these proteins have been char- acterized by cloning and sequence analysis of the corres- ponding cDNA. Some properties of the macromolecules have

  17. Sequence-dependent conformational heterogeneity of a hybrid DNA.RNA dodecamer duplex.

    PubMed

    Gao, X; Jeffs, P W

    1994-05-01

    Two- and three-dimensional homonuclear NMR studies of a hybrid duplex RI, formed by annealing r(GCGCAAAACGCG) and d(CGCGTTTTGCGC) strands are described. NMR parameters, such as intra- and interresidue proton-proton NOEs and sugar proton coupling constants were analyzed with reference to those of the corresponding DNA.DNA duplex. Furthermore, spectral analyses were conducted on the basis of model structures of nucleic acid duplexes. Distinctive spectral patterns of the hybrid duplex reveal unique heterogeneous conformations which co-exist throughout the sequence and are significantly different from those of model structures of either canonical A- or B-forms. Features of an intermediate conformation were observed in the DNA and RNA strands in duplex RI, the former being more B-like and the latter more A-like. Three-dimensional NOESY-NOESY spectra were analyzed and their use was demonstrated for resolving superimposed resonances and cross peaks, especially those originating from the RNA strand. The application of a useful strategy that combines the use of 2D NMR data and the known structural information for efficient 3D spectral analyses is demonstrated. PMID:7517242

  18. DNA/RNA transverse current sequencing: intrinsic structural noise from neighboring bases

    PubMed Central

    Alvarez, Jose R.; Skachkov, Dmitry; Massey, Steven E.; Kalitsov, Alan; Velev, Julian P.

    2015-01-01

    Nanopore DNA sequencing via transverse current has emerged as a promising candidate for third-generation sequencing technology. It produces long read lengths which could alleviate problems with assembly errors inherent in current technologies. However, the high error rates of nanopore sequencing have to be addressed. A very important source of the error is the intrinsic noise in the current arising from carrier dispersion along the chain of the molecule, i.e., from the influence of neighboring bases. In this work we perform calculations of the transverse current within an effective multi-orbital tight-binding model derived from first-principles calculations of the DNA/RNA molecules, to study the effect of this structural noise on the error rates in DNA/RNA sequencing via transverse current in nanopores. We demonstrate that a statistical technique, utilizing not only the currents through the nucleotides but also the correlations in the currents, can in principle reduce the error rate below any desired precision.

  19. Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA

    NASA Astrophysics Data System (ADS)

    Reichl, Maren; Herzog, Mario; Greiss, Ferdinand; Wolff, Manuel; Braun, Dieter

    2015-06-01

    Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012), 10.1073/pnas.1215764109], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis.

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

    PubMed Central

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

    2012-01-01

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

  1. Differentiation of strains of tick-borne encephalitis virus by means of RNA-DNA hybridization.

    PubMed

    Shamanin, V A; Pletnev, A G; Rubin, S G; Zlobin, V I

    1990-07-01

    Cloned cDNA and synthetic deoxyoligonucleotides, complementary to various parts of the genomic RNA of tick-borne encephalitis virus (TBEV), strain Sofjin, were used to distinguish between strains of TBEV and other flaviviruses. The cDNA probe hybridized with strains of TBEV and related flaviviruses of the TBE complex except for Powassan virus, and it did not react with flaviviruses of the Japanese encephalitis and dengue subgroups. Viruses of the TBE complex and some strains of TBEV were differentiated from TBEV strain Sofjin by the thermal stability of RNA-DNA hybrids. Negishi and louping-ill viruses were the most closely related to TBEV strain Sofjin, among viruses of the TBE complex. Eight strains of TBEV isolated in different geographical areas from different sources were tested by dot-hybridization with 11 deoxyoligonucleotide probes. The probes revealed genetic variations among strains of TBEV. The pattern of hybridization correlated with the source of virus strains: TBEV strains isolated from TBE patients reacted with more probes than strains isolated from ticks. Within a group of epidemic strains of TBEV there was a correlation between the geographical area of isolation and similarity to TBEV strain Sofjin. PMID:2374007

  2. Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism.

    PubMed

    Jurkowski, Tomasz P; Meusburger, Madeleine; Phalke, Sameer; Helm, Mark; Nellen, Wolfgang; Reuter, Gunter; Jeltsch, Albert

    2008-08-01

    Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism. PMID:18567810

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

    PubMed Central

    Schwartz, Chad; Guo, Peixuan

    2013-01-01

    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

  4. 24-Hour Rhythms of DNA Methylation and Their Relation with Rhythms of RNA Expression in the Human Dorsolateral Prefrontal Cortex

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Collagen-DNA complex.

    PubMed

    Svintradze, David V; Mrevlishvili, George M; Metreveli, Nunu; Jariashvili, Ketevan; Namicheishvili, Luisa; Skopinska, Joana; Sionkowska, Alina

    2008-01-01

    Previously presented models of collagen-DNA (7) and collagen-siRNA (8) complexes point to a general description of delivery systems and indicate to what specific topology that system should be equipped with to effectively deliver the gene into the living body via in vivo and in vitro injection. We focused our interest on the nature of collagen-DNA complex structure and the molecular and environmental determinants of the self-association processes of collagen with the presence of DNA. In this aspect, the self-association of collagen-DNA complex offers an opportunity to characterize a unique system, which may be related to the general mechanisms of self-association of fiber macromolecules by water bridges. For characterizing the collagen-DNA interaction, we used FTIR-ATR, NMR, and AFM experiments done on a separate collagen film, DNA film, and on the peptide-DNA aqueous solution. We demonstrate that collagen-DNA spontaneously forms self-assembling complex systems in aqueous solution. Such self-association of the complex could be induced by electrostatic interactions between neutral collagen cylinders, having strong dipole moment, and negatively charged DNA cylinders. A final complex could be formed by hydrogen bonds between specified donor groups of collagen and phosphate acceptor groups of DNA. According to FTIR measurements, a collagen triple helix should not change global conformation during collagen-DNA complex formation. PMID:18052128

  7. A comparative study of COI and 16 S rRNA genes for DNA barcoding of cultivable carps in India.

    PubMed

    Mohanty, Mausumee; Jayasankar, Pallipuram; Sahoo, Lakshman; Das, Paramananda

    2015-02-01

    The 5' region of the mitochondrial DNA gene cytochrome c oxidase subunit I (COI) is the standard marker for DNA barcoding. However, 16?S rRNA has also been advocated for DNA barcoding in many animal species. Herein, we directly compare the usefulness of COI and 16?S rRNA in discriminating six cultivable carp species: Labeo rohita, Catla catla, Cirrhinus mrigala, Labeo fimbriatus, Labeo bata and Cirrhinus reba from India. Analysis of partial sequences of these two gene fragments from 171 individuals indicated close genetic relationship between Catla catla and Labeo rohita. The results of the present study indicated COI to be more useful than 16?S rRNA for DNA barcoding of Indian carps. PMID:24047160

  8. MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells.

    PubMed

    Xie, Chen; Chen, Wei; Zhang, Mengdie; Cai, Qiuxian; Xu, Weiyi; Li, Xiaodi; Jiang, Songshan

    2015-07-01

    Despite extensive investigation into the role of let-7 miRNAs in pathological tumor processes, their involvement in the DNA damage response remains unclear. Here we show that most let-7 family members down-regulate MDM4 expression via binding to MDM4 mRNA at a conserved DNA sequence. Expression of exogenous let-7 miRNA mimics decreased MDM4 protein but not mRNA levels. Several DNA damage reagents increased let-7 expression, thereby decreasing MDM4 protein levels in glioma cells. Inhibition of endogenous let-7 with antisense RNAs rescued MDM4 protein levels with or without MG132, a proteasome-dependent degradation inhibitor. An MDM4 mutation identified in a glioma patient was associated with loss of the putative MDM4 target site. Therefore, let-7 binding to MDM4 is implicated in the DNA damage response. PMID:26028311

  9. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    SciTech Connect

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  10. Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

    PubMed Central

    Miller, C A; Patterson, W L; Johnson, P K; Swartzell, C T; Wogoman, F; Albarella, J P; Carrico, R J

    1988-01-01

    A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses. PMID:2457597

  11. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication.

    PubMed Central

    Hatton, T; Zhou, S; Standring, D N

    1992-01-01

    The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible. Images PMID:1501273

  12. Differential utility of the Bacteroidales DNA and RNA markers in the tiered approach for microbial source tracking in subtropical seawater.

    PubMed

    Liu, Rulong; Cheng, Ken H F; Wong, Klaine; Cheng, Samuel C S; Lau, Stanley C K

    2015-07-01

    Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater. PMID:25652655

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

    PubMed Central

    Pang, Bo; McFaline, Jose L.; Burgis, Nicholas E.; Dong, Min; Taghizadeh, Koli; Sullivan, Matthew R.; Elmquist, C. Eric; Cunningham, Richard P.; Dedon, Peter C.

    2012-01-01

    Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism. PMID:22308425

  14. The Origins of the RNA World

    PubMed Central

    Robertson, Michael P; Joyce, Gerald F

    2012-01-01

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

  15. Characterization of a Viroid-Derived RNA Promoter for the DNA-Dependent RNA Polymerase from Escherichia coli †

    Microsoft Academic Search

    Martin Pelchat; Catherine Grenier; Jean-Pierre Perreault

    2002-01-01

    This paper attributes a novel function, namely, that of transcriptional promoter, to the self- complementary, self-cleaving hammerhead RNA sequences found in RNA derived from the peach latent mosaic viroid (PLMVd). The features of this RNA promoter, which adopts a hairpin structure that can be utilized by Escherichia coli RNA polymerase (RNAP) for in vitro transcription, that trigger the RNAP driven

  16. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    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…

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

    E-print Network

    Hedges, Blair

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

  18. DUPLEX: A molecular mechanics program in torsion angle space for computing structures of DNA and RNA

    SciTech Connect

    Hingerty, B.E.

    1992-07-01

    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.

  19. Simplified Identification of mRNA or DNA in Whole Cells

    NASA Technical Reports Server (NTRS)

    Almeida, Eduardo; Kadambi, Geeta

    2007-01-01

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

  20. In-vitro nanodiagnostic platform through nanoparticles and DNA-RNA nanotechnology.

    PubMed

    Chan, Ki; Ng, Tzi Bun

    2015-04-01

    Nanocomposites containing nanoparticles or nanostructured domains exhibit an even higher degree of material complexity that leads to an extremely high variability of nanostructured materials. This review introduces analytical concepts and techniques for nanomaterials and derives recommendations for a qualified selection of characterization techniques for specific types of samples, and focuses the characterization of nanoparticles and their agglomerates or aggregates. In addition, DNA nanotechnology and the more recent newcomer RNA nanotechnology have achieved almost an advanced status among nanotechnology researchers¸ therefore, the core features, potential, and significant challenges of DNA nanotechnology are also highlighted as a new discipline. Moreover, nanobiochips made by nanomaterials are rapidly emerging as a new paradigm in the area of large-scale biochemical analysis. The use of nanoscale components enables higher precision in diagnostics while considerably reducing the cost of the platform that leads this review to explore the use of nanoparticles, nanomaterials, and other bionanotechnologies for its application to nanodiagnostics in-vitro. PMID:25761622

  1. Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

    Microsoft Academic Search

    Suzette A. Pabit; Xiangyun Qiu; Jessica S. Lamb; Li Li; Steve P. Meisburger; Lois Pollack

    2009-01-01

    The recent discovery of the RNA interference mech- anism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we pres- ent a comparative investigation of electrostatic effects in RNA and

  2. A Non-Heme Iron-Mediated Chemical Demethylation in DNA and RNA

    PubMed Central

    Yi, Chengqi; Yang, Cai-Guang; He, Chuan

    2010-01-01

    CONSPECTUS DNA methylation is arguably one of the most important chemical signals in biology. However, aberrant DNA methylation can lead to cytotoxic or mutagenic consequences. A DNA repair protein in Escherichia coli, AlkB, corrects some of the unwanted methylations of DNA bases by a unique oxidative demethylation in which the methyl carbon is liberated as formaldehyde. The enzyme also repairs exocyclic DNA lesions—that is, derivatives in which the base is augmented with an additional heterocyclic subunit—by a similar mechanism. Two proteins in humans that are homologous to AlkB, ABH2 and ABH3, repair the same spectrum of lesions; another human homologue of AlkB, FTO, is linked to obesity. In this Account, we describe our studies of AlkB, ABH2, and ABH3, including our development of a general strategy to trap homogeneous protein–DNA complexes through active-site disulfide cross-linking. AlkB uses a non-heme mononuclear iron(II) and the cofactors 2-ketoglutarate (2KG) and dioxygen to effect oxidative demethylation of the DNA base lesions 1-methyladenine (1-meA), 3-methylcytosine (3-meC), 1-methylguanine (1-meG), and 3-methylthymine (3-meT). ABH3, like AlkB, works better on single-stranded DNA (ssDNA) and is capable of repairing damaged bases in RNA. Conversely, ABH2 primarily repairs lesions in double-stranded DNA (dsDNA); it is the main housekeeping enzyme that protects the mammalian genome from 1-meA base damage. The AlkB-family proteins have moderate affinities for their substrates and bind DNA in a non-sequence-specific manner. Knowing that these proteins flip the damaged base out from the duplex DNA and insert it into the active site for further processing, we first engineered a disulfide cross-link in the active site to stabilize the Michaelis complex. Based on the detailed structural information afforded by the active-site cross-linked structures, we can readily install a cross-link away from the active site to obtain the native-like structures of these complexes. The crystal structures show a distinct base-flipping feature in AlkB and establish ABH2 as a dsDNA repair protein. They also provide a molecular framework for understanding the demethylation reaction catalyzed by these proteins and help to explain their substrate preferences. The chemical cross-linking method demonstrated here can be applied to trap other labile protein–DNA interactions and can serve as a general strategy for exploring the structural and functional aspects of base-flipping proteins. PMID:19852088

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

    PubMed Central

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

    2014-01-01

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

  4. Structure-based methods for the phylogenetic analysis of ribosomal RNA molecules 

    E-print Network

    Gillespie, Joseph James

    2005-11-01

    Ribosomal RNA (rRNA) molecules form highly conserved secondary and tertiary structures via rRNA-rRNA and rRNA-protein interactions that collectively comprise the macromolecule that is the ribosome. Because of their cellular ...

  5. A Functional Interface at the rDNA Connects rRNA Synthesis, Pre-rRNA Processing and Nucleolar Surveillance in Budding Yeast

    PubMed Central

    Leporé, Nathalie; Lafontaine, Denis L. J.

    2011-01-01

    Ribogenesis is a multistep error-prone process that is actively monitored by quality control mechanisms. How ribosomal RNA synthesis, pre-rRNA processing and nucleolar surveillance are integrated is unclear. Nor is it understood how defective ribosomes are recognized. We report in budding yeast that, in vivo, the interaction between the transcription elongation factor Spt5 and Rpa190, the largest subunit of RNA polymerase (Pol) I, requires the Spt5 C-terminal region (CTR), a conserved and highly repetitive domain that is reminiscent of the RNA Pol II C-terminal domain (CTD). We show that this sequence is also required for the interaction between Spt5 and Nrd1, an RNA specific binding protein, and an exosome cofactor. Both the Spt4-Spt5, and the Nrd1-Nab3 complexes interact functionally with Rrp6, and colocalize at the rDNA. Mutations in the RNA binding domain of Nrd1, but not in its RNA Pol II CTD-interacting domain, and mutations in the RRM of Nab3 led to the accumulation of normal and aberrant polyadenylated pre-rRNAs. Altogether these results indicate that Nrd1-Nab3 contributes to recruiting the nucleolar surveillance to elongating polymerases to survey nascent rRNA transcripts. PMID:21949810

  6. In-situ crosslinking hydrogels for combinatorial delivery of chemokines and siRNA-DNA carrying microparticles to dendritic cells

    PubMed Central

    Singh, Ankur; Suri, Shalu; Roy, Krishnendu

    2009-01-01

    Polymer-based, injectable systems that can simultaneously deliver multiple bioactive agents in a controlled manner could significantly enhance the efficacy of next generation therapeutics. For immunotherapies to be effective, both prophylactically or therapeutically, it is not only critical to drive the antigen (Ag) specific immune response strongly towards either T helper type 1 (Th1) or Th2 phenotype, but also to promote recruitment of a high number of antigen-presenting cells (APCs) at the site of immunization. We have recently reported a microparticle-based system capable of simultaneously delivering siRNA and DNA to APCs. Here we present an in situ crosslinkable, injectable formulation containing dendritic cell (DC)-chemoattractants and dual-mode DNA-siRNA loaded microparticles to attract immature DCs and simultaneously deliver, to the migrated cells, immunomodulatory siRNA and plasmid DNA antigens. These low crosslink density hydrogels were designed to degrade within 2–7 days in-vitro and released chemokines in a sustained manner. Chemokine carrying gels attracted 4–6 folds more DCs over a sustained period in vitro, compared to an equivalent bolus dose. Interestingly, migrated DCs were able to infiltrate the hydrogels and efficiently phagocytose the siRNA/DNA carrying microparticles. Hydrogel embedded microparticles co-delivering Interleukin-10 siRNA and plasmid DNA antigens exhibited efficient Interleukin-10 gene knockdown in migrated primary DCs in-vitro. PMID:19560815

  7. Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis

    PubMed Central

    Ahmed, Ikhlak; Sarazin, Alexis; Bowler, Chris; Colot, Vincent; Quesneville, Hadi

    2011-01-01

    Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ?26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H?=?A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5? to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression. PMID:21586580

  8. Reliability of T7Based mRNA Linear Amplification Validated by Gene Expression Analysis of Human Kidney Cells Using cDNA Microarrays

    Microsoft Academic Search

    Michael Rudnicki; Susanne Eder; Gabriele Schratzberger; Bernd Mayer; Timothy W. Meyer; Martin Tonko; Gert Mayer

    2004-01-01

    Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp® amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and

  9. DNA sequence, structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA from Podospora anserina

    Microsoft Academic Search

    Donald J. Cummings; Joanne M. Domenico; James Nelson; Mitchell L. Sogin

    1989-01-01

    Summary DNA sequence analysis and the localization of the 5? and 3? termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli

  10. Genetic Evidence That DNA Methyltransferase DRM2 Has a Direct Catalytic Role in RNA-Directed DNA Methylation in Arabidopsis thaliana

    PubMed Central

    Naumann, Ulf; Daxinger, Lucia; Kanno, Tatsuo; Eun, Changho; Long, Quan; Lorkovic, Zdravko J.; Matzke, Marjori; Matzke, Antonius J. M.

    2011-01-01

    RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic modification in plants. We report here the identification of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) in a forward screen for mutants defective in RdDM in Arabidopsis thaliana. The finding of a mutation in the presumptive active site argues in favor of direct catalytic activity for DRM2. PMID:21212233

  11. Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor

    PubMed Central

    Shu, Dan; Khisamutdinov, Emil F.; Zhang, Le; Guo, Peixuan

    2014-01-01

    Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ?18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ?18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies. PMID:24084081

  12. Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

    PubMed

    Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

    2015-02-01

    Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells. PMID:25579461

  13. Stretching Helical Macromolecules

    NASA Astrophysics Data System (ADS)

    Carri, Gustavo A.

    2005-03-01

    We study the elasticity of a homopolypeptide under extension using Monte Carlo simulations based on the Wang-Landau algorithm. The effect of external mechanical forces is described with an extension of a model for helical polymers (V. Varshney et. al., Macromolecules 2004, 37, 8794). We find that the application of a mechanical force first increases the helix-to-coil transition temperature and then decreases it. This non-monotonic behavior is a consequence of a change in the nature of the helix-coil transition which becomes a helix-extended-coil transition for strong forces. We also find that the force-elongation curve at constant temperature displays three different behaviors depending on the temperature of the system. At temperatures below or slightly above the helix-coil transition temperature the force-elongation curve shows one or two coexistence regions, respectively. In these regions helical sequences and random coil domains coexist. At high temperatures our model recovers the elastic behavior of a random coil. We present a quantitative comparison with the theoretical results of Buhot and Halperin, and very good agreement is observed.

  14. DNA-RNA hybrids contribute to the replication dependent genomic instability induced by Omcg1 deficiency.

    PubMed

    Houlard, Martin; Artus, Jérôme; Léguillier, Teddy; Vandormael-Pournin, Sandrine; Cohen-Tannoudji, Michel

    2011-01-01

    During S phase, the replisome has to overcome many physical obstacles that can cause replication fork stalling and compromise genome integrity. Transcription is an important source of replicative stress and consequently, maintenance of genome integrity requires the protection of chromosomes from the deleterious effects arising from the interaction between nascent RNAs and template DNA, leading to stable DNA-RNA hybrids (R-loop) formation. We previously reported the essential role of Omcg1 (Ovum Mutant Candidate Gene) for cell cycle progression during early embryonic development. Here, we show that OMCG1 is a target of the cell cycle checkpoint kinases ATR/ATM and is essential for S phase progression in mouse embryonic fibroblasts. Using a conditional gene inactivation strategy, we demonstrate that OMCG1 depletion impairs cell viability as a consequence of DSB formation, checkpoint activation and replication fork collapse. We also show that no chromosome breaks were generated in non-cycling Omcg1-deficient cells. Furthermore, increased RNaseH expression significantly alleviated genomic instability in deficient fibroblasts suggesting that cotranscriptional R-loops formation contributes to the genesis of replication-dependent DSBs in these cells. Together with recent reports describing its participation to complexes involved in cotanscriptional processes, our results suggest that OMCG1 plays a role in the tight coupling between mRNA processing pathways and maintenance of genome integrity during cell cycle progression. PMID:21191184

  15. Increased Mitochondrial DNA and RNA Polymerase Activity in Ethylene-Treated Potato Tubers

    PubMed Central

    Apelbaum, Akiva; Vinkler, Chana; Sfakiotakis, Evangalos; Dilley, David R.

    1984-01-01

    A purified mitochondrial fraction was isolated from potato (Solanum tuberosum L.) tubers respiring normally at 23°C or at an accelerated rate in response to treatment with ethylene (10 microliters per liter). A pronounced increase in various mitochondrial enzymic activities was observed in response to exposure of the whole tubers to ethylene. Cytochrome c oxidase activity increased more than 50%, DNA polymerase activity increased about 2-fold, and RNA polymerase activity increased 2.5-fold. Moreover, DNA or RNA polymerase activities of mitochondria isolated from tubers not treated with ethylene were not affected by ethylene treatment in vitro. Respiratory control ratios decreased from 2.84 to 1.50 with increasing periods of ethylene treatment from 0 to 15 hours. None of these changes were observed in untreated tubers. It is concluded that the stimulation of respiration by ethylene in potato tubers is accompanied in vivo by an enhancement of mitochondrial enzymic activity of both membrane-associated enzymes which participate in the mitochondrial oxidative electron transport as well as soluble enzymes which are not directly involved in respiration. PMID:16663865

  16. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules.

    PubMed

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50-1000?bp) within 50?s, a mixture of protein molecules (20-340?kDa) within 5?s, and a mixture of RNA molecules (100-1000?bases) within 25?s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

  17. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    PubMed Central

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000?bp) within 50?s, a mixture of protein molecules (20–340?kDa) within 5?s, and a mixture of RNA molecules (100–1000?bases) within 25?s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

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

    PubMed Central

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

    2013-01-01

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

  19. Mass isotopomer analysis of nucleosides isolated from RNA and DNA using GC/MS.

    PubMed

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

    2015-01-01

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

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

    Microsoft Academic Search

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

    1983-01-01

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

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

    PubMed Central

    Patterson, Jennifer; Mura, Cameron

    2013-01-01

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

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

    PubMed Central

    Arima, Hidetoshi; Motoyama, Keiichi; Higashi, Taishi

    2012-01-01

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

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA

    SciTech Connect

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

    1991-04-01

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

  6. Modeling of Antigenomic Therapy of Mitochondrial Diseases by Mitochondrially Addressed RNA Targeting a Pathogenic Point Mutation in Mitochondrial DNA*

    PubMed Central

    Tonin, Yann; Heckel, Anne-Marie; Vysokikh, Mikhail; Dovydenko, Ilya; Meschaninova, Mariya; Rötig, Agnès; Munnich, Arnold; Venyaminova, Alya; Tarassov, Ivan; Entelis, Nina

    2014-01-01

    Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene. PMID:24692550

  7. Effect of intercalator substituent and nucleotide sequence on the stability of DNA- and RNA-naphthalimide complexes.

    PubMed

    Johnson, Charles A; Hudson, Graham A; Hardebeck, Laura K E; Jolley, Elizabeth A; Ren, Yi; Lewis, Michael; Znosko, Brent M

    2015-07-01

    DNA intercalators are commonly used as anti-cancer and anti-tumor agents. As a result, it is imperative to understand how changes in intercalator structure affect binding affinity to DNA. Amonafide and mitonafide, two naphthalimide derivatives that are active against HeLa and KB cells in vitro, were previously shown to intercalate into DNA. Here, a systematic study was undertaken to change the 3-substituent on the aromatic intercalator 1,8-naphthalimide to determine how 11 different functional groups with a variety of physical and electronic properties affect binding of the naphthalimide to DNA and RNA duplexes of different sequence compositions and lengths. Wavelength scans, NMR titrations, and circular dichroism were used to investigate the binding mode of 1,8-naphthalimide derivatives to short synthetic DNA. Optical melting experiments were used to measure the change in melting temperature of the DNA and RNA duplexes due to intercalation, which ranged from 0 to 19.4°C. Thermal stabilities were affected by changing the substituent, and several patterns and idiosyncrasies were identified. By systematically varying the 3-substituent, the binding strength of the same derivative to various DNA and RNA duplexes was compared. The binding strength of different derivatives to the same DNA and RNA sequences was also compared. The results of these comparisons shed light on the complexities of site specificity and binding strength in DNA-intercalator complexes. For example, the consequences of adding a 5'-TpG-3' or 5'-GpT-3' step to a duplex is dependent on the sequence composition of the duplex. When added to a poly-AT duplex, naphthalimide binding was enhanced by 5.6-11.5°C, but when added to a poly-GC duplex, naphthalimide binding was diminished by 3.2-6.9°C. PMID:25960324

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

    PubMed Central

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

    2014-01-01

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

  9. Nucleotide sequence of the region between the 18S rRNA sequence and the 28S rRNA sequence of rat ribosomal DNA.

    PubMed Central

    Subrahmanyam, C S; Cassidy, B; Busch, H; Rothblum, L I

    1982-01-01

    The DNA sequence of the intragenic region of the rat 45S ribosomal RNA precursor was determined. This sequence contains 2282 nucleotides and extends from the conserved EcoR I site near the 3' terminus of 18S rRNA to 69 nucleotides downstream of the 5' terminus of 28S rRNA. The sequences corresponding to 18S and 5.8S rRNA were identified by comparison with previously published data. The 5' terminus of rat 28S rRNA was identified by S1 nuclease protection and reverse transcriptase elongation assays. The internal transcribed spacers were found to be 1066 and 765 nucleotides long and had little homology with those of Xenopus and yeast. Regions of sequence homology between rat and Xenopus were found at the junctions of the internal transcribed spacers with 18S, 5.8S and 28S rRNA. These homologies suggest that these sequences may function as recognition sites for the processing of the ribosomal precursor RNA. Images PMID:6287418

  10. Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton

    Microsoft Academic Search

    Jorg Peplies; Stanley Chun Kwan Lau; Jakob Pernthaler; Rudolf Amann; Frank Oliver Glockner

    2004-01-01

    Summary An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior ampli- fication by polymerase chain reaction (PCR). The results obtained were compared with those of quan- titative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially

  11. Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells? †

    PubMed Central

    Bellecave, Pantxika; Cazenave, Christian; Rumi, Julie; Staedel, Cathy; Cosnefroy, Ophélie; Andreola, Marie-Line; Ventura, Michel; Tarrago-Litvak, Laura; Astier-Gin, Thérèse

    2008-01-01

    We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection. PMID:18347106

  12. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

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

    PubMed

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

    2015-02-18

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

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

    PubMed Central

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

    2015-01-01

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

  15. Targeting of A701G nucleotide at the human ATP1A1 locus using a RNA/DNA chimera.

    PubMed

    Cervelli, Tiziana; Lombardi, Grazia; Citti, Lorenzo; Galli, Alvaro; Locci, Maria Teresa; Rainaldi, Giuseppe

    2002-01-01

    The single base substitution mediated by chimeric RNA/DNA oligonucleotide is a new promising approach of gene therapy for single base mutation diseases. We exploited this approach to render HeLa cells resistant to ouabain by introducing a single base substitution in the alpha 1 subunit of the NA+/K+ ATPase human gene. The chimeric oligonucleotide was administered to HeLa cells by electroporation and the frequency of ouabain resistant cells determined. The results showed that the chimeric RNA/DNA oligonucleotide failed to enhance the frequency of ouabain resistant cells supporting the controversy about the conflicting results of the technique. PMID:12537020

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

    SciTech Connect

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

    2008-12-01

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

  17. Direct-methods determination of an RNA/DNA hybrid decamer at 1.15 A resolution.

    PubMed

    Han, G W

    2001-02-01

    For the first time, ab initio direct methods have been used to solve the crystal structure of an RNA/DNA hybrid decamer. The RNA/DNA sequence corresponds to the leftmost two-thirds of the polypurine tract (PPT), the primer for second-strand DNA synthesis by HIV-1 reverse transcriptase (RT). Direct methods using Shake-and-Bake (SnB) yielded solutions for the RNA/DNA decamer molecule using 1.15 A data, which is just on the resolution edge of what might work with direct methods. Atomic positions for 96% of the entire molecule, containing 514 non-H atoms including three Ca(2+) ions, were easily interpreted from a Fourier map based on the 'Shake-and-Bake' minimal function and CROQUE phase-refinement program. Only six atoms, primarily in the sugar linkage, were missing in this Fourier map. At present, the R factor of the model is 0.143 (R(free) = 0.186) for the 562 non-H atom sites located. The conformation of the RNA/DNA helix is A-form, with a typical A-helix minor-groove width. This paper presents the methodology used in solving this structure. PMID:11173466

  18. Chronic progressive external ophthalmoplegia: A new heteroplasmic tRNA Leu(CUN) mutation of mitochondrial DNA

    Microsoft Academic Search

    E. Cardaioli; P. Da Pozzo; E. Malfatti; G. N. Gallus; A. Rubegni; A. Malandrini; C. Gaudiano; L. Guidi; G. Serni; G. Berti; M. T. Dotti; A. Federico

    2008-01-01

    We sequenced all genes of mitochondrial tRNAs of a patient with chronic progressive external ophthalmoplegia with 5% ragged red fibres and 15% COX-negative fibres but without macrorearrangements of mitochondrial DNA (mtDNA). Direct sequencing showed a novel heteroplasmic G>A substitution in position 12316 of tRNALeu(CUN) gene. This change destroys a highly conserved G–C base coupling in tRNA T?C branch. By RFLP

  19. Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with H218O

    PubMed Central

    Rettedal, Elizabeth A; Brözel, Volker S

    2015-01-01

    Current limitations in culture-based methods have lead to a reliance on culture-independent approaches, based principally on the comparative analysis of primary semantides such as ribosomal gene sequences. DNA can be remarkably stable in some environments, so its presence does not indicate live bacteria, but extracted ribosomal RNA (rRNA) has previously been viewed as an indicator of active cells. Stable isotope probing (SIP) involves the incorporation of heavy isotopes into newly synthesized nucleic acids, and can be used to separate newly synthesized from existing DNA or rRNA. H218O is currently the only potential universal bacterial substrate suitable for SIP of entire bacterial communities. The aim of our work was to compare soil bacterial community composition as revealed by total versus SIP-labeled DNA and rRNA. Soil was supplemented with H218O and after 38 days the DNA and RNA were co-extracted. Heavy nucleic acids were separated out by CsCl and CsTFA density centrifugation. The 16S rRNA gene pools were characterized by DGGE and pyrosequencing, and the sequence results analyzed using mothur. The majority of DNA (?60%) and RNA (?75%) from the microcosms incubated with H218O were labeled by the isotope. The analysis indicated that total and active members of the same type of nucleic acid represented similar community structures, which suggested that most dominant OTUs in the total nucleic acid extracts contained active members. It also supported that H218O was an effective universal label for SIP for both DNA and RNA. DNA and RNA-derived diversity was dissimilar. RNA from this soil more comprehensively recovered bacterial richness than DNA because the most abundant OTUs were less numerous in RNA than DNA-derived community data, and dominant OTU pools didn't mask rare OTUs as much in RNA. PMID:25650291

  20. DNA complementary to parathyroid mRNA directs synthesis of pre-proparathyroid hormone in a linked transcription-translation system

    Microsoft Academic Search

    Henry M. Kronenberg; Bryan E. Roberts; Joel F. Habener; John T. Potts; Alexander Rich

    1977-01-01

    DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription-translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal Sequencing and by electrophoretic and immunologic criteria.

  1. Current Cancer Drug Targets, 2001, 1, 241-247 241 D-RNAi (Messenger RNA-antisense DNA Interference) as a Novel

    E-print Network

    Chuong, Cheng-Ming

    Current Cancer Drug Targets, 2001, 1, 241-247 241 D-RNAi (Messenger RNA-antisense DNA Interference, CA 90033, USA Abstract: D-RNAi (Messenger RNA-antisense DNA interference), a novel transduction of -catenin D- RNAi was shown to knock out more than 99% endogenous -catenin gene expression

  2. Evaluation of novel carbon nano-tube particles in the bacterial and viral DNA and RNA extraction from the clinical samples

    Microsoft Academic Search

    Nguyen KC; Vo DXA; Hoang HN; Ho LTT; Pham HV

    2010-01-01

    Molecular techniques have become the most im- portant methods of detecting bacterial and viral pathogens. However, current genomic extraction methods are currently limited in term of automation. In this study, carbon nano-tube was used as the vector to trap DNA and RNA molecules. The capability of carbon nano-tube to trap DNA and RNA was evaluated using samples (TB and HBV

  3. The structure of an RNA/DNA hybrid: a substrate of the ribonuclease activity of HIV-1 reverse transcriptase.

    PubMed

    Horton, N C; Finzel, B C

    1996-12-01

    The structure of a complementary hybrid duplex of RNA and DNA has been determined by X-ray crystallography. A ten residue DNA oligonucleotide of sequence 5'-G-G-C-G-C-C-C-G-A-A-3' was annealed to complementary RNA (5'-u-u-c-g-g-g-c-g-c-c-3') and crystallized, producing tetragonal crystals that diffract to 2.3 A resolution. The hybrid adopts a geometry that is neither strictly A nor B-form, rather the helix possesses qualities of both, reminiscent of spectroscopic descriptions of a hybrid conformation, or H-form. All of the ribonucleotides maintain the C3'-endo conformation seen in A-form, while both C3'-endo and C2'-endo conformations are found in the deoxyribonucleotides. The minor groove width (8.5 to 10.5 A) is intermediate between standard values for A (11 A) and B-form (7.4 A) DNA. The global parameters rise and base-pairs tilt (or inclination) are like that of A-DNA, however the slide and x displacement (Dx) are more like that of A-RNA, thus giving the hybrid a unique conformation. In addition, the 10-mer crystallizes in a manner that allows the formation of dimers that stack end-to-end, thereby providing a glimpse of how an extended (20 base-pair) helix of RNA-DNA hybrid might appear. This duplex sequence was selected for study because it is specifically recognized by the ribonuclease H function of HIV reverse transcriptase. A structure of a substrate of this enzyme is of potential value in understanding requirements for the selectivity of this important drug target. The minor groove of the hybrid duplex, lined with the 2-OH of the ribose rings, is the single distinguishing characteristic of the RNA/DNA hybrid, undoubtedly an important structural feature conferring selectivity. PMID:8969302

  4. Hybrid Opto-electric Manipulation of Macromolecules

    NASA Astrophysics Data System (ADS)

    Wereley, Steve

    2013-11-01

    Recently our research group has developed an innovative method for capturing, concentrating, manipulating and sorting populations of micro- and nanometer-scaled entities-particles, cells, macro-molecules, etc. These populations range from individual particles to thousands of particles (Lab-on-a-Chip, 2008; Microfluidics and Nanofluidics, 2008) while the sizes range from microns to nanometers. This novel technique combines features of optical trapping and dielectrophoresis in an innovative, dynamic way using a simple parallel plate electrode configuration. Transparent electrodes comprised of Indium Tin Oxide (ITO) on glass substrates are used to generate an electric field in the fluid while at the same time allowing light into and out of the fluid. Near-IR optical illumination causes subtle localized heating, creating an electric permittivity gradient that in turn drives a microscopic toroidal vortex. The vortex efficiently transports particles to a preferred location, usually the surface of the electrode. Recent advances have extended have allowed us to apply this technique to macromolecules (DNA, proteins) as well as nanoscale particles (quantum dots, nanowires and PSL particles).

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

    SciTech Connect

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

    2011-09-15

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

  6. A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.

    PubMed Central

    Kambhampati, S

    1995-01-01

    Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID:7534409

  7. A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.

    PubMed

    Kambhampati, S

    1995-03-14

    Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID:7534409

  8. A sequence-specific interaction between the Saccharomyces cerevisiae rRNA gene repeats and a locus encoding an RNA polymerase I subunit affects ribosomal DNA stability.

    PubMed

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

    2015-02-01

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

  9. [The differentiation of viruses of the tick-borne encephalitis complex by means of RNA-DNA hybridization].

    PubMed

    Shamanin, V A; Pletnev, A G; Rubin, S G; Zlobin, V I

    1991-01-01

    Nucleic acid spot hybridization with cloned cDNA of tick-borne encephalitis (TBE) virus, strain Sofjin, was used to differentiate strains of TBE and other flaviviruses. The cDNA probe reacted with strains of TBE and flaviviruses of TBE subgroup with the exception of Powassan virus. The probe did not react with viruses of Japanese encephalitis and Gendue subgroups. The viruses of TBE subgroup and some strains of TBE virus were differentiated from TBE strain Sofjin by thermal stability of RNA-DNA hybrids. Negishi and Louping ill viruses were found to be most closely related to TBE strain Sofjin among viruses of the TBE subgroup. PMID:1713371

  10. MACROMOLECULES, Part 1* Carbohydrates, Lipids, and Nucleic Acids

    E-print Network

    Prestwich, Ken

    MACROMOLECULES, Part 1* Carbohydrates, Lipids, and Nucleic Acids Introduction: Living organisms are unique in being composed of long, massive molecules called macromolecules. Biochemistry, the study of macromolecules: carbohydrates, lipids, proteins and nucleic acids. All macromolecules are polymers (chains

  11. INFLUENCE OF MACROMOLECULES ON CHEMICAL TRANSPORT

    EPA Science Inventory

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

  12. RNA/DNA ratios in American glass eels (Anguilla rostrata): evidence for latitudinal variation in physiological status

    E-print Network

    Bernatchez, Louis

    RNA/DNA ratios in American glass eels (Anguilla rostrata): evidence for latitudinal variation Oc´eans, 850 Route de la Mer, Mont-Joli, QC, G5H 3ZH, Canada Keywords Eel, fish, migration hatched American eels undergo an extensive oceanic migration from the Sargasso Sea toward coastal

  13. Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

    Microsoft Academic Search

    Megan J. Wilson; Iain L. Lamont

    2006-01-01

    The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in

  14. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA

    Microsoft Academic Search

    Mary Barbara; Marie Gu; Meredith Gibson; Carolyn Modi; Neil Winegarden; Norman N. Iscove

    2002-01-01

    Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification

  15. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

    PubMed

    Lever, Mark A; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  16. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    PubMed Central

    Lever, Mark A.; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

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

    PubMed Central

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

    2013-01-01

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

  18. Soluble Interleukin-6 Receptor-Mediated Innate Immune Response to DNA and RNA Viruses

    PubMed Central

    Wang, Qing; Chen, Xueyuan; Feng, Jian; Cao, Yanhua; Song, Yu; Wang, Hui; Zhu, Chengliang; Liu, Shi

    2013-01-01

    The interleukin-6 (IL-6) receptor, which exists as membrane-bound and soluble forms, plays critical roles in the immune response. The soluble IL-6 receptor (sIL6R) has been identified as a potential therapeutic target for preventing coronary heart disease. However, little is known about the role of this receptor during viral infection. In this study, we show that sIL6R, but not IL-6, is induced by viral infection via the cyclooxygenase-2 pathway. Interestingly, sIL6R, but not IL-6, exhibited extensive antiviral activity against DNA and RNA viruses, including hepatitis B virus, influenza virus, human enterovirus 71, and vesicular stomatitis virus. No synergistic effects on antiviral action were observed by combining sIL6R and IL-6. Furthermore, sIL6R mediated antiviral action via the p28 pathway and induced alpha interferon (IFN-?) by promoting the nuclear translocation of IFN regulatory factor 3 (IRF3) and NF-?B, which led to the activation of downstream IFN effectors, including 2?,5?-oligoadenylate synthetase (OAS), double-stranded RNA-dependent protein kinase (PKR), and myxovirus resistance protein (Mx). Thus, our results demonstrate that sIL6R, but not IL-6, plays an important role in the host antiviral response. PMID:23946454

  19. 'Effects of Elevated Temperature on Dehalococcoides Dechlorination Performance and DNA and RNA Biomarker Abundance

    SciTech Connect

    Fletcher, Kelly E [ORNL; Costanza, Jed [University of Tennessee; Cruz-Garcia, Claribel [University of Tennessee; Ramaswamy, Nivedhya [University of Tennessee; Pennell, Kurt [University of Tennessee; Loeffler, Frank E [ORNL

    2011-01-01

    Coupling thermal treatment with microbial reductive dechlorination is a promising remedy for tetrachloroethene (PCE) and trichloroethene (TCE) contaminated source zones. Laboratory experiments evaluated Dehalococcoides (Dhc) dechlorination performance, viability, and biomarker gene (DNA) and transcript (mRNA) abundances during exposure to elevated temperatures. The PCE-dechlorinating consortia BDI and OW produced ethene when incubated at temperatures of 30 C, but vinyl chloride (VC) accumulated when cultures were incubated at 35 or 40 C. Cultures incubated at 40 C for less than 49 days resumed VC dechlorination following cooling; however, incubation at 45 C resulted in complete loss of dechlorination activity. Dhc 16S rRNA, bvcA, and vcrA gene abundances in cultures showing complete dechlorination to ethene at 30 C exceeded those measured in cultures incubated at higher temperatures, consistent with observed dechlorination activities. Conversely, biomarker gene transcript abundances per cell in cultures incubated at 35 and 40 C were generally at least one order-of-magnitude greater than those measured in ethene-producing cultures incubated at 30 C. Even in cultures accumulating VC, transcription of the vcrA gene, which is implicated in VC-to-ethene dechlorination, was up-regulated. Temperature stress caused the up-regulation of Dhc reductive dehalogenase gene expression indicating that Dhc gene expression measurements should be interpreted cautiously as Dhc biomarker gene transcript abundances may not correlate with dechlorination activity.

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

    PubMed

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

    1989-07-01

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

  1. Chemical Modification Patterns of Active and Inactive as Well as Procapsid-Bound and Unbound DNA-Packaging RNA of Bacterial Virus Phi29

    Microsoft Academic Search

    Chunlin Zhang; Mark Trottier; Chaoping Chen; Peixuan Guo

    2001-01-01

    During replication, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into the limited space within the preformed procapsid. We previously found that a viral-encoded RNA (pRNA) played a key role in bacterial virus phi29 DNA translocation. Design of mutant pRNA sets containing two and three inactive mutant pRNAs, respectively, led to the conclusion that the stoichiometry of

  2. Biology, Macromolecules 1 1 Biology 120 Textbook Reading Assignment: (for Macromolecules 2 -

    E-print Network

    Prestwich, Ken

    Biology, Macromolecules 1 1 Biology 120 Textbook Reading Assignment: (for Macromolecules 2 - Proteins) Sadava Ch. 3, Sections 3.1 and 3.2 General: The notes packet (Macromolecules 2) for this section;Biology, Macromolecules 1 2 1. Describe the four levels of protein structure. Explain the importance

  3. Intro Biology, Macromolecules 1 1 Biology 131 Textbook Reading Assignment: (for Macromolecules 1)

    E-print Network

    Prestwich, Ken

    Intro Biology, Macromolecules 1 1 Biology 131 Textbook Reading Assignment: (for Macromolecules 1: macromolecule dehydration chitin purine monomer hydrolysis fat pyrimidine polymer hexose sterol ribose isomer and nucleic acids and understand the role each type of macromolecule plays in the cell · understand

  4. Dominant and specific repression of Xenopus oocyte 5S RNA genes and satellite I DNA by histone H1.

    PubMed Central

    Wolffe, A P

    1989-01-01

    The genome of Xenopus laevis contains two large families of class III genes (oocyte 5S RNA and satellite I DNA) that are repressed in somatic cells. Both gene families are actively transcribed in a soluble extract of X.laevis oocyte nuclei, using chromatin deficient in histone H1 as a template. The addition of histone H1, to this transcriptionally active chromatin, results in the dominant and selective repression of oocyte 5S RNA genes and satellite I DNA. Somatic 5S RNA genes remain active following histone H1 addition. Changes in chromatin structure could have a dominant role in the regulation of class III gene expression during Xenopus embryogenesis. Images PMID:2721490

  5. Structure and dynamics of DNA and RNA oligonucleotides as studied using solution and solid state NMR. [NMR (nuclear magnetic resonance)

    SciTech Connect

    Wang, A.C.C.

    1992-01-01

    NMR experiments reveal that the base H8/H6 and H1[prime] protons of RNA have T[sub 1] relaxation times that are distinctly longer than those of DNA. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. Results from solid state [sup 2]H NMR experiments on the purine C-8 deuterium-labeled 12 base pair RNA duplex [r(CG*CG*A*A*UUGG*CG*)][sub 2] were compared with results obtained by other investigators on the 12 base pair DNA duplex [d(CG*CG*A*A*TTCG*CG*)][sub 2]. The motional amplitudes of DNA and RNA purines are similar at 0%-88% RH and their internal rates of motion are different at 0%-80% RH. The assumption that dodecameric oligonucleotides (12-mers) tumble isotropically in solution is often used when calculating proton-proton distances from NOE data. The authors have undertaken the task of testing the isotropic assumption using experimental NMR data. The authors have calculated the structure of [d(GCGTTTAAACGC)][sub 2] using both the isotropic assumption and the assumption that the duplex tumbles anisotropically in solution like a perfect cylinder. The resulting structures from both approaches are virtually indistinguishable. The isotropic assumption is valid for oligonucleotides 12 base pairs and shorter. The solution structure of the 12 base pair hybrid chimeric duplex [r(gcg)d(TATATACGC)][sub 2] has been solved using NMR techniques combined with distance geometry and NOE back-calculation methods. The structure is characterized by a dramatic bend of 52[degrees] in the helix axis. The location of the bend is not at the RNA-DNA step but occurs between the first and second residues of the DNA segment. The center of the DNA TATATA segment has a remarkably narrow minor groove that becomes very wide in the hybrid portions of the duplex.

  6. DNA Damage activates A Spatially Distinct Late Cytoplasmic Cell Cycle Checkpoint Network Controlled by MK2-mediated RNA Stabilization

    PubMed Central

    Reinhardt, H. Christian; Hasskamp, Pia; Schmedding, Ingolf; Morandell, Sandra; van Vugt, Marcel .A.T.M.; Wang, XiaoZhe; Linding, Rune; Ong, Shao-En; Weaver, David; Carr, Steven A.

    2010-01-01

    Summary Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G2/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of post-transcriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2, phosphorylates hnRNPA0, to stabilize Gadd45? mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45? mRNA degradation. Gadd45? functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the post-transcriptional regulation of gene expression as part of the DNA damage response in cancer cells. PMID:20932473

  7. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization.

    PubMed

    Reinhardt, H Christian; Hasskamp, Pia; Schmedding, Ingolf; Morandell, Sandra; van Vugt, Marcel A T M; Wang, Xiaozhe; Linding, Rune; Ong, Shao-En; Weaver, David; Carr, Steven A; Yaffe, Michael B

    2010-10-01

    Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45? mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45? mRNA degradation. Gadd45? functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells. PMID:20932473

  8. Missing Genes, Multiple ORFs, and C-to-U Type RNA Editing in Acrasis kona (Heterolobosea, Excavata) Mitochondrial DNA

    PubMed Central

    Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G.E.; Baldauf, Sandra L.

    2014-01-01

    Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. PMID:25146648

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-11-10

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

  11. RESEARCH ARTICLES Analytical Shape Computation of Macromolecules

    E-print Network

    Dai, Yang

    RESEARCH ARTICLES Analytical Shape Computation of Macromolecules: I. Molecular Area and Volume properties of macromolecules based on the alpha shape theory. This method uses the duality between alpha- cepts behind the alpha shape theory and the algorithm for computing areas and volumes of macromolecules

  12. RNA-targeted activators, but not DNA-targeted activators, repress the synthesis of short transcripts at the human immunodeficiency virus type 1 long terminal repeat.

    PubMed Central

    Pendergrast, P S; Hernandez, N

    1997-01-01

    The human immunodeficiency virus type 1 (HIV-1) promoter directs the synthesis of two types of RNA molecules: full-length transcripts, whose synthesis is activated by the viral activator Tat, and short transcripts, whose synthesis is dependent on the inducer of short transcripts (IST), a bipartite DNA element located in large part downstream of the HIV-1 transcriptional start site. In the absence of Tat, short transcripts constitute the large majority of the RNA molecules synthesized from the HIV-1 promoter. In the presence of Tat, synthesis of the short transcripts is repressed and synthesis of the full-length transcripts is activated. Tat is unique among transcriptional activators in acting through an RNA target, the TAR element. However, Tat has been shown to activate transcription from a DNA target when fused to the appropriate DNA binding domain, raising the question of why Tat has been directed to the RNA. Here we have compared the abilities of Tat and other RNA- and DNA-bound activators to stimulate transcription from the HIV-1 promoter. We show that DNA-targeted activators, including DNA-targeted Tat, activate the synthesis of both short and long transcripts, while RNA-targeted Tat and another RNA-targeted activator activate the synthesis of full-length transcripts but specifically repress that of short transcripts. The unique ability of RNA-targeted activators to down-regulate short transcript synthesis suggests that Tat is directed to the RNA specifically for the purpose of repressing short transcripts. PMID:8995607

  13. Cloning and sequencing of cDNA encoding the human ribosomal protein L11 mRNA

    SciTech Connect

    Mishin, V.P.; Filipenko, M.L.; Muravlev, A.I. [Novosibirsk Inst. of Bioorganic chemistry (Russian Federation)] [and others

    1995-02-01

    To clone the RPL11 cDNA, we used a polymerase chain reaction (PCR) with the single-stranded cDNA synthesized on the total placentary poly(A){sup +}mRNA with the use of primer M245 containing a 3{prime}-terminal oligo(dT)-tract, the 5{prime}terminal hexadecanucleotide sequence of the M13 universal primer, and a NotiI restriction site between them. On the basis of the known sequence of the 5{prime}-end of the human ribosomal protein L11 mRNA, we chose two partially overlapping deoxyribooligonucleotides as 5{prime}-terminal primers in the amplification of the RPL11 cDNA. A pair of partially overlapping oligonucleotides complementary to the oligo(dT)-containing primer were used as 3{prime}-terminal primers.

  14. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system.

    PubMed Central

    Sasaki, Y F; Ayusawa, D; Oishi, M

    1994-01-01

    We report a novel procedure to construct a normalized (equalized) cDNA library. By introduction of the highly efficient self-hybridization system between a whole mRNA population and their corresponding cDNA immobilized on latex beads, which involves relatively simple manipulations, we were able to generate an mRNA population in which the copy number of abundant species was reduced while that of rare species was enriched. In a typical experiment, after several cycles of self-hybridization on the beads, the ratio of the most to the least abundant marker mRNA species dropped by a factor of 300 (from 10,000 to 30) while the complexity and length of mRNAs in the population remained unchanged. The procedure should provide a potent tool for the expression cloning of cDNA and also facilitate the construction of whole cDNA catalogs from specific tissues (or cell types) from higher organisms. Images PMID:8152931

  15. The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence.

    PubMed

    Yukl, Steven A; Shergill, Amandeep K; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K; Crouch, Pierre; Deeks, Steven G; Havlir, Diane V; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K

    2013-10-15

    Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir. PMID:23852128

  16. The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

    PubMed Central

    Yukl, Steven A.; Shergill, Amandeep K.; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K.; Crouch, Pierre; Deeks, Steven G.; Havlir, Diane V.; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K.

    2013-01-01

    Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir. PMID:23852128

  17. The Ability to Form Homodimers Is Essential for RDM1 to Function in RNA-Directed DNA Methylation

    PubMed Central

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

    2014-01-01

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

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

    Microsoft Academic Search

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

    2001-01-01

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

  19. 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)

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

    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.

  20. cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides.

    PubMed

    Chen, Rong; Li, Wensheng; Lin, Haoran

    2005-09-01

    A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper. PMID:16005251

  1. Distribution of DNA and RNA content in the bone marrow and alveolar macrophages of rats after subchronic inhalation of formaldehyde

    SciTech Connect

    Dallas, C.E.; Mellard, D.N.; Theiss, J.C.; Pentecost, A.R.; Fairchild, E.J. II

    1987-06-01

    The technique of flow cytometry was used to monitor the cell-cycle distribution of DNA and RNA in selected tissues of rats that were subchronically exposed to formaldehyde (HCHO) inhalation. Male Sprague Dawley rats inhaled HCHO vapor concentrations of 0, 0.5, 3, or 15 ppm for 6 hr/day, 5 days/week, for up to 24 weeks. Simultaneous two-parameter measurements were made on a Phywe ICP 22 pulse cytophotometer by use of an acridine orange stain of the DNA and RNA of each cell sampled. No significant changes relative to the controls were determined in the percentage S and G/sub 2/ + M phases of the DNA from simple tissues of the HCHO-exposed animals. Increases of 50 to 60% in the RNA content of G/sub 1/ cells (RI) in the alveolar macrophages were seen after 1 week of exposure at all three HCHO doses. This effect was diminished after subchronic exposure. No HCHO-related effect was observed, though, in the RI of the rat bone marrow cells at any time point. However, the observed changes in RNA content were of rather limited magnitude relative to the cell-cycle perturbation induced by known cytotoxic agents in this and other flow cytometry studies. These modest and transient RI increases therefore probably did not reflect any significant effect on cell viability or cell proliferation in the affected tissues.

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

    PubMed Central

    Taira, Naoe; Yamaguchi, Tomoko; Kimura, Junko; Lu, Zheng-Guang; Fukuda, Shinji; Higashiyama, Shigeki; Ono, Masaya; Yoshida, Kiyotsugu

    2014-01-01

    Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage. PMID:24379358

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

    Microsoft Academic Search

    Ruth Nussinov; Haim J. Wolfson

    1991-01-01

    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

  4. DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

    PubMed

    Andrade-Lima, Leonardo C; Veloso, Artur; Paulsen, Michelle T; Menck, Carlos F M; Ljungman, Mats

    2015-03-11

    The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases. PMID:25722371

  5. DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes

    PubMed Central

    Andrade-Lima, Leonardo C.; Veloso, Artur; Paulsen, Michelle T.; Menck, Carlos F.M.; Ljungman, Mats

    2015-01-01

    The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5?-3? direction with slow recovery and TC-NER at the 3? end of long genes. RNA synthesis resumed fully at the 3?-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases. PMID:25722371

  6. Nature-inspired DNA nanosensor for real-time in situ detection of mRNA in living cells.

    PubMed

    Tay, Chor Yong; Yuan, Liang; Leong, David Tai

    2015-05-26

    Rapid and precise in situ detection of gene expressions within a single cell is highly informative and offers valuable insights into its state. Detecting mRNA within single cells in real time and nondestructively remains an important challenge. Using DNA nanotechnology and inspired by nature's many examples of "protective-yet-accessible" exoskeletons, we designed our mRNA nanosensor, nano-snail-inspired nucleic acid locator (nano-SNEL), to illustrate these elements. The design of the nano-SNEL is composed of a sensory molecular beacon module to detect mRNA and a DNA nanoshell component, mimicking the functional anatomy of a snail. Accurate and sensitive visualization of mRNA is achieved by the exceptional protection conferred by the nanoshell to the sensory component from nucleases-mediated degradation by approximately 9-25-fold compared to its unprotected counterpart. Our nano-SNEL design strategy improved cell internalization is a demonstration of accurate, dynamic spatiotemporal resolved detection of RNA transcripts in living cells. PMID:25906327

  7. RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus: a zinc metalloenzyme.

    PubMed

    Auld, D S; Kawaguchi, H; Livingston, D M; Vallee, B L

    1974-05-01

    RNA tumor viruses contain a characteristic RNA-dependent DNA polymerase (reverse transcriptase) which has been thought to be related to the induction of leukemia by this virus. A disturbance in a zinc-dependent enzyme system was first postulated to account for the demonstrated differences in zinc metabolism of normal and leukemic leukocytes [Vallee et al. in (1949) Acta Unio. Int. Contra Cancrum 6, 869 and (1950) Acta Unio. Int. Contra Cancrum 6, 1102]. In order to investigate the relationship between zinc and the initiation of leukemia in chickens by avian myeloblastosis virus, we have examined the metalloenzyme nature of its reverse transcriptase. The present data show that this protein is a zinc metalloenzyme demonstrating the postulated relationship between zinc and a leukemic process. Paucity of purified enzyme generated the design of a novel system of analysis incorporating microwave-induced emission spectrometry combined with gel exclusion chromatography. It provides precision, reproducibility, and remarkable limits of detection on mul samples containing 10(-12) to 10(-14) g-atoms of metal, and is thus orders of magnitude more sensitive than other methods. The chromatographic fraction with highest enzymatic activity contains 1.8 x 10(-11) g-atoms of zinc per 1.6 mug of protein, corresponding to either 1.8 or 2.0 g-atoms of zinc per mole of enzyme for a molecular weight previously determined either as 1.6 or 1.8 x 10(5). Copper, iron and manganese are absent, i.e., at or below the limits of detection, 10(-13) to 10(-14) g-atoms. Agents known to chelate zinc inhibit the enzyme, while their nonchelating isomers do not. The data underline the participation of zinc in nucleic acid metabolism and bear importantly upon the lesions that accompany leukemia and zinc deficiency. PMID:4134617

  8. Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents

    PubMed Central

    Tournier, E.; Amenc, L.; Pablo, A.L.; Legname, E.; Blanchart, E.; Plassard, C.; Robin, A.; Bernard, L.

    2015-01-01

    An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents. The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. • A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters. • The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult. • The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

  9. Mitochondrial DNA of the sea anemone, Metridium senile (Cnidaria): Prokaryote-like genes for tRNA f-Met and small-subunit ribosomal RNA, and standard genetic code specificities for AGR and ATA codons

    Microsoft Academic Search

    Geneviève A. Pont-Kingdon; C. Timothy Beagley; Ronald Okimoto; David R. Wolstenholme

    1994-01-01

    The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the sea anemone Metridium senile (phylum Cnidaria, class Anthozoa, order Actiniaria) has been determined, within which have been identified the genes for respiratory chain NADH dehydrogenase subunit 2 (ND2), the small-subunit rRNA (s-rRNA), cytochrome c oxidase subunit II(COII), ND4, ND6, cytochrome b (Cyt b), tRNAf-Met, and the

  10. External field influence on semiflexible macromolecules: geometric coupling

    E-print Network

    Stefano Bellucci; Yevgeny Mamasakhlisov; Armen Nersessian

    2011-03-22

    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.

  11. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli.

    PubMed

    Perez-Rodriguez, Ritsdeliz; Haitjema, Charles; Huang, Qingqiu; Nam, Ki Hyun; Bernardis, Sarah; Ke, Ailong; DeLisa, Matthew P

    2011-02-01

    A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR-associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR-Cas system was activated in cells expressing a plasmid-encoded protein that was targeted to the twin-arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two-component regulatory system, which is known to respond to extracytoplasmic stress. Furthermore, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR-Cas system targets DNA directly as part of a defence mechanism in bacteria that is overlapping with but not limited to phage infection. PMID:21255106

  12. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli

    PubMed Central

    Perez-Rodriguez, Ritsdeliz; Haitjema, Charles; Huang, Qingqiu; Nam, Ki Hyun; Bernardis, Sarah; Ke, Ailong; DeLisa, Matthew P.

    2011-01-01

    SUMMARY A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR-associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR-Cas system was activated in cells expressing a plasmid-encoded protein that was targeted to the twin-arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two-component regulatotory system, which is known to respond to extracytoplasmic stress. Further, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR-Cas system targets DNA directly as part of a defense mechanism in bacteria that is overlapping with but not limited to phage infection. PMID:21255106

  13. High Variety of Known and New RNA and DNA Viruses of Diverse Origins in Untreated Sewage

    PubMed Central

    Ng, Terry Fei Fan; Marine, Rachel; Wang, Chunlin; Simmonds, Peter; Kapusinszky, Beatrix; Bodhidatta, Ladaporn; Oderinde, Bamidele Soji; Wommack, K. Eric

    2012-01-01

    Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, <10?4), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses. PMID:22933275

  14. Mitochondrial DNA heteroplasmy dynamics in a kindred harboring a novel pathogenic mutation in the mitochondrial tRNA glutamate gene

    SciTech Connect

    Moraes, C.T.; Hao, H. [Univ. of Miami, FL (United States); Bonilla, E.; DiMauro, S.

    1994-09-01

    We have identified a novel mitochondrial DNA (mtDNA) mutation in a 32-year-old male with a myopathy (without progressive external ophthalmoplegia) and mild pyramidal involvement. This A{yields}G transition at mtDNA position 14709 alters an evolutionary conserved nucleotide in a region coding for the anticodon loop of the mitcohondrial tRNA{sup Glu}. The 14709 mtDNA mutation was heteroplasmic but present at very high levels in the patient`s muscle (95%), white blood cells (81%) and hair follicles (90%). The same mutant mtDNA population was observed in white blood cells and hair follicles of all maternal relatives, but a lesser percentage (25-80%). The patient`s muscle showed many ragged-red fibers and a severe focal defect in cytochrome c oxidase activity, accompanied by the absence of cross-reacting material for mitochondrially synthesized polypeptides (ND 1 and COX II). The percentage of mutant mtDNA was not preferentially increased over two generations. Rather, the percentage of mutant mtDNA observed in siblings seemed to follow a normal distribution around the percentage observed in their mothers. Single hair PCR/RFLP analysis showed that the intercellular fluctuation in the percentage of mutant mtDNA differs among family members. Younger generations tend to have a more homogeneous distribution of mutant mtDNA in different hair follicles. The highest degree of variability between individual hair follicles was observed in the patient`s grandmother. These results suggest that the intercellular distribution of the mutant and wild-type mtDNA populations may drift towards homogeneity in subsequent generations.

  15. Nonempirically Tuned Range-Separated DFT Accurately Predicts Both Fundamental and Excitation Gaps in DNA and RNA Nucleobases

    PubMed Central

    2012-01-01

    Using a nonempirically tuned range-separated DFT approach, we study both the quasiparticle properties (HOMO–LUMO fundamental gaps) and excitation energies of DNA and RNA nucleobases (adenine, thymine, cytosine, guanine, and uracil). Our calculations demonstrate that a physically motivated, first-principles tuned DFT approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many-body GW theory. Furthermore, in the same set of nucleobases, we show that the nonempirical range-separated procedure also leads to significantly improved results for excitation energies compared to conventional DFT methods. The present results emphasize the importance of a nonempirically tuned range-separation approach for accurately predicting both fundamental and excitation gaps in DNA and RNA nucleobases. PMID:22904693

  16. Computational Insights into the Interactions between DNA and siRNA with “Rigid” and “Flexible” Triazine Dendrimers

    PubMed Central

    Pavan, Giovanni M.; Mintzer, Meredith A.; Simanek, Eric E.; Merkel, Olivia M.; Kissel, Thomas; Danani, Andrea

    2013-01-01

    In this study, simulation challenges intuitive models of “flexible” and “rigid” generation two triazine dendrimers as it pertains to solution conformation and conformation on binding DNA or siRNA sequences. These results derive from structural and energetic analyses of the binding events. Simulations of the rigid structure reinforce the role of the constrained piperazine linker in positioning the peripheral groups at significant distance from each other and the core of the dendrimer. In contrast, the flexible dendrimer, characterized by triethyleneglycol-like linkers, collapses in solution. On binding DNA and siRNA, these conformations are largely retained. The rigid dendrimer undergoes reorganization of peripheral groups to generate a large number of contacts to the nucleic acid. In contrast, the flexible dendrimer, originally conceived to create multivalent interactions with nucleic acids, generates only a few contacts and collapses further. This paper provides unique insight in the role played by molecular flexibility in the binding phenomenon. PMID:20131771

  17. Development of Targeted Recombinant Polymers that can deliver siRNA to the Cytoplasm and Plasmid DNA to the Cell Nucleus

    PubMed Central

    Canine, Brenda F.; Wang, Yuhua; Ouyang, Wenyun; Hatefi, Arash

    2011-01-01

    One of the major limitations to effective siRNA delivery is the lack of a siRNA-specific delivery system. Currently, the same delivery systems that are used for plasmid DNA (pDNA) delivery to the cell nucleus are used for siRNA delivery to the cytoplasm. To fill this gap, the objective of this study was to design a biopolymer that can be programmed via its amino acid sequence to deliver siRNA specifically to cytoplasm. For pDNA delivery, a nuclear localization signal (NLS) was added to the biopolymer structure to facilitate active translocation of the genetic material towards nucleus. The biopolymers were complexed with pEGFP and GFP-siRNA and used to transfect SKOV-3 (HER2+) cells. The intracellular trafficking of the nanoparticles was also monitored in real-time and live cells. The results demonstrated that the biopolymer with NLS is a suitable carrier for pDNA delivery but not siRNA delivery. Conversely, the biopolymer without NLS was suitable for siRNA delivery to the cytoplasm but not pDNA to the cell nucleus. The potential use of the designed biopolymer for combination therapy of cancer cells with gene (thymidine kinase) and siRNA (BCL2) was also examined in SKOV-3 cancer cells. PMID:21192992

  18. A novel heteroplasmic tRNA Leu(CUN) mtDNA point mutation associated with chronic progressive external ophthalmoplegia

    Microsoft Academic Search

    Elena Cardaioli; Paola Da Pozzo; Elena Radi; Maria Teresa Dotti; Antonio Federico

    2005-01-01

    We have sequenced all mitochondrial tRNA genes from a patient with chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy, who had no detectable large mtDNA deletions. Direct sequencing failed to detect previously reported mutations and showed a heteroplasmic mutation at nucleotide 12,276 in the tRNALeu(CUN) gene, in the dihydrouridine stem, which is highly conserved through the species during evolution. RFLP

  19. The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods.

    PubMed

    Yan, Fengting; Wu, Xin; Crawford, Melissa; Duan, Wenrui; Wilding, Emily E; Gao, Li; Nana-Sinkam, S Patrick; Villalona-Calero, Miguel A; Baiocchi, Robert A; Otterson, Gregory A

    2010-12-01

    Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR. PMID:20888418

  20. RNA–DNA ratio and other nucleic acid-based indicators for growth and condition of marine fishes

    Microsoft Academic Search

    L. Buckley; E. Caldarone; T.-L. Ong

    1999-01-01

    Nucleic acid analysis has provided useful tools to study recent growth and mortality of young fishes and their responses to environmental variability. The ratio of RNA–DNA (R\\/D) has been shown to respond to changes in feeding conditions and growth after periods as short as 1–3 days in a variety of fish species. The earliest studies used primarily UV-based methods, but

  1. TARGETING OF A701G NUCLEOTIDE AT THE HUMAN ATP1A1 LOCUS USING A RNA\\/DNA CHIMERA

    Microsoft Academic Search

    Tiziana Cervelli; Grazia Lombardi; Lorenzo Citti; Alvaro Galli; Maria Teresa Locci; Giuseppe Rainaldi

    2002-01-01

    The single base substitution mediated by chimeric RNA\\/DNA oligonucleotide is a new promising approach of gene therapy for single base mutation diseases. We exploited this approach to render HeLa cells resistant to ouabain by introducing a single base substitution in the alpha 1 subunit of the NA\\/K ATPase human gene. The chimeric oligonucleotide was administered to HeLa cells by electroporation

  2. The solution structure of a DNA·RNA duplex containing 5-propynyl U and C; comparison with 5Me modifications

    Microsoft Academic Search

    Jeffrey I. Gyi; Daquan Gao; Graeme L. Conn; John O. Trent; Tom Brown

    The addition of the propynyl group at the 5 position of pyrimidine nucleotides is highly stabilising. We have determined the thermodynamic stability of the DNA·RNA hybrid r(GAAGAGAAGC)·d(GCpUpUpCpUp- CpUpUpC) where p is the propynyl group at the 5 position and compared it with that of the unmodified duplex and the effects of methyl substitutions. The incorporation of the propyne group at

  3. Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

    Microsoft Academic Search

    Nicolas Carrasco; Yuri Buzin; Elizabeth Tyson; Elizer Halpert; Zhen Huang

    2004-01-01

    We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2¢-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2¢-methylseleno cytidine phosphoramidite and improved the accessibility of the 2¢-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into

  4. Effects of trace metals on growth of yellow perch ( Perca flavescens ) as measured by RNA-DNA ratios

    Microsoft Academic Search

    Paul K. Kearns; Gary J. Atchison

    1979-01-01

    Synopsis Relationships between sublethal concentrations of cadmium and zinc in natural water and metal uptake by and growth of fish were investigated. RNA-DNA ratios and weight gain were used to assess seasonal growth differences between yellow perch populations from contaminated and control sites. Whole-body concentrations of cadmium and zinc in young-of-the-year perch (Perca flavescens) were significantly different between sites. Measurable

  5. The Search for an Optimal DNA, RNA, and Protein Detection by in situ Hybridization, Immunohistochemistry, and Solution-Based Methods

    PubMed Central

    Yan, Fengting; Wu, Xin; Crawford, Melissa; Duan, Wenrui; Wilding, Emily E; Gao, Li; Nana-Sinkam, S. Patrick; Villalona-Calero, Miguel A; Baiocchi, Robert A.; Otterson, Gregory A.

    2010-01-01

    Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and Bovine Papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was “gentle” fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as “gentle” cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or -if one does not have unfixed tissues - solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR. PMID:20888418

  6. Separate quality-control measures are necessary for estimation of RNA and methylated DNA from formalin-fixed, paraffin-embedded specimens by quantitative PCR.

    PubMed

    Korlimarla, Aruna; Prabhu, Jyothi S; Anupama, C E; Remacle, Jose; Wahi, Kanu; Sridhar, T S

    2014-03-01

    Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's ?. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA. PMID:24412525

  7. Studies of RNA and DNA hairpin interactions with mineral surface: implications for molecules in meteorites and Martian samples

    NASA Astrophysics Data System (ADS)

    El Amri, S.; Grajcar, L.; Fermandjian, S.; Ghomi, M.; Baron, M. H.; Maurel, M. C.

    2001-08-01

    In previous studies, we have shown that Surface-Enhanced Spectroscopy (SERS) using silver colloids as adsorbent phase allows studying picomoles of DNA or RNA nucleic acids standing at the solid-liquid interface. The most important underlined feature was the use of the adenyl groups as probes of nucleic acid reactivity with minerals. In this report, we present the silver phase as a model for electron-depleted mineral surfaces that may be encountered in terrestrial soils. In a first approach, we focus on the adsorption of adenyl residues included in four species of DNA loops with three residues or in the eight GNRA RNA tetraloops. They could be considered as in vitro models to test nucleic acid adsorption mechanisms and also to enlighten potential structural and reactivity changes that may be entailed. Our study underlines that primary sequences and local structures monitor the reactivity of adenyl residues involved in loops, whether made of DNA or RNA. Such investigations on very small amount of nucleic acid materials could serve in the area of searching living molecules adsorbed on long live terrestrial materials or in extraterrestrial rocks.

  8. Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

    PubMed Central

    Yang, Ching-Fu; Chen, Kuan-Chun; Cheng, Ying-Hui; Raja, Joseph A. J.; Huang, Ya-Ling; Chien, Wan-Chu; Yeh, Shyi-Dong

    2014-01-01

    Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. PMID:25030413

  9. Systems based mapping demonstrates that recovery from alkylation damage requires DNA repair, RNA processing, and translation associated networks

    PubMed Central

    Rooney, John P.; George, Ajish D.; Patil, Ashish; Begley, Ulrike; Bessette, Erin; Zappala, Maria R.; Huang, Xin; Conklin, Douglas A.; Cunningham, Richard P.; Begley, Thomas J.

    2009-01-01

    The identification of cellular responses to damage can promote mechanistic insight into stress signalling. We have screened a library of 3,968 E. coli gene deletion mutants to identify 99 gene products that modulate the toxicity of the alkylating agent methyl methanesulfonate (MMS). We have developed an ontology mapping approach to identify functional categories over-represented with MMS-toxicity modulating proteins and demonstrate that, in addition to DNA re-synthesis (replication, recombination, and repair), proteins involved in mRNA processing and translation influence viability after MMS damage. We have also mapped our MMS-toxicity modulating proteins onto an E. coli protein interactome and identified a sub-network consisting of 32 proteins functioning in DNA repair, mRNA processing, and translation. Clustering coefficient analysis identified seven highly connected MMS-toxicity modulating proteins associated with translation and mRNA processing, with the high connectivity suggestive of a coordinated response. Corresponding results from reporter assays support the idea that the SOS response is influenced by activities associated with the mRNA-translation interface. PMID:18824089

  10. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

  11. Site-selective cleavage of structured RNA by a staphylococcal nuclease-DNA hybrid.

    PubMed Central

    Zuckermann, R N; Schultz, P G

    1989-01-01

    A hybrid enzyme consisting of an oligodeoxyribonucleotide fused to a unique site on staphylococcal nuclease site-selectively cleaves a number of natural RNAs including Escherichia coli M1 RNA (377 bases), 16S rRNA (1542 bases), and yeast tRNA(Phe). The oligonucleotide directs the nuclease activity of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. In the case of M1 RNA, hydrolysis occurs primarily at one phosphodiester bond, converting 50% of the starting material to product. Furthermore, the reaction products can be enzymatically manipulated: tRNA(Phe) was cleaved in the anticodon region and was religated to form the full-length tRNA in high yield. Because the specificity of these hybrid enzymes can be easily altered, they should prove to be useful tools for probing RNA structure and function. Images PMID:2467291

  12. DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity

    PubMed Central

    Cao, Mengmeng; Wei, Candong; Zhao, Lili; Wang, Jingfeng; Jia, Qiannan; Wang, Xue

    2014-01-01

    ABSTRACT The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed of the PB1, PB2, and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nuclei of infected cells. In this study, we newly identified host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with the bPB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, while the J domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to enter the nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner. IMPORTANCE The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaptation. In this study, we newly identified a host protein, DnaJA1/Hsp40, that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nuclei of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into the nucleus along with the nuclear import of the PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, which is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of the Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs. PMID:25253355

  13. HBV-Specific shRNA is Capable of Reducing the Formation of Hepatitis B Virus Covalently Closed Circular DNA, but has No Effect on Established Covalently Closed Circular DNA in vitro

    PubMed Central

    Starkey, Jason L.; Chiari, Estelle F.; Isom, Harriet C.

    2009-01-01

    Summary Hepatitis B virus (HBV) covalently closed circular DNA (CCC DNA) is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA) expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication and the levels of HBV RNA, HBsAg, replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced through day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA to antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication. PMID:19088280

  14. Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)

    PubMed Central

    Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E.; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-01-01

    DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

  15. USING A COMMERCIAL DNA EXTRACTION KIT TO OBTAIN RNA FROM MATURE RICE KERNELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraction of total RNA from starchy plant material such as common food grains is difficult, and especially so from dry rice (Oryza sativa L.) kernels. Most commercial RNA kits are not suited for starchy materials and traditional RNA extraction procedures leave hazardous organic wastes that have ex...

  16. Preparation of cDNA Library for dRNA-seq

    Technology Transfer Automated Retrieval System (TEKTRAN)

    microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide ...

  17. Enhanced Radiation and Chemotherapy-mediated Cell Killing of Human Cancer Cells by Small Inhibitory RNA Silencing of DNA Repair Factors1

    Microsoft Academic Search

    Spencer J. Collis; Michael J. Swartz; William G. Nelson; Theodore L. DeWeese

    2003-01-01

    Recent developments in the use of small inhibitory RNA molecules (siRNAs) to inhibit specific protein expression have highlighted the po- tential use of siRNA as a therapeutic agent. The double-strand break signaling\\/repair proteins ATM, ATR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are attractive targets to confer enhanced radio and chemosensitivity to tumor cells. We have designed and exog- enously

  18. Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA)

    Microsoft Academic Search

    Albert Heim; Isabella Maria Grumbach; Stefanie Zeuke; Bert Top

    1998-01-01

    NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription\\/ polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron

  19. Segment-specific expression of alkaline phosphatase in the Tubifex embryo requires DNA replication and mRNA synthesis.

    PubMed

    Kitamura, Kaoru; Shimizu, Takashi

    2002-04-15

    During embryogenesis of the oligochaete annelid Tubifex, segments VII and VIII specifically express mesodermal alkaline phosphatase (ALP) activity in the ventrolateral region. In this study, using specific inhibitors, we examined whether this segment-specific expression of ALP activity depends on DNA replication and RNA transcription. BrdU-incorporation experiments showed that presumptive ALP-expressing cells undergo the last round of DNA replication at 12-24 hr prior to emergence of ALP activity. When this DNA replication was inhibited by aphidicolin, ALP development was completely abrogated in the ventrolateral mesoderm. Similar inhibition of ALP development was also observed in alpha-amanitin-injected embryos. While injection of alpha-amanitin at 24 hr prior to the emergence of ALP activity exerted inhibitory effects on ALP development, injection at 14 hr was no longer effective. In contrast, ALP activity developed normally in cytochalasin-D-treated embryos in which cytokinesis was prevented from occurring for 36 hs prior to appearance of ALP activity. These results suggest that the segment-specific development of ALP activity in the Tubifex embryo depends on DNA replication and mRNA transcription, both of which occur long before the emergence of ALP activity. PMID:11932950

  20. Strand-Biased Formation of G-Quadruplexes in DNA Duplexes Transcribed with T7 RNA Polymerase.

    PubMed

    Liu, Jia-Quan; Xiao, Shan; Hao, Yu-Hua; Tan, Zheng

    2015-07-27

    G-quadruplex-forming sequences are enriched near transcription start sites (TSSs) in animal genes. They readily form G-quadruplexes in transcription, which in turn regulate transcription. Therefore, the control of G-quadruplex formation is important for their functionality. It is now shown that G-quadruplexes form efficiently on the non-template, but hardly on the template DNA strand in the downstream vicinity of TSSs in DNA duplexes when they are transcribed by the T7 RNA polymerase (RNAP). Structural analysis reveals that the T7 RNAP causes distortion in a DNA duplex both inside and in front of the enzyme. This structural distortion leads to strand-biased G-quadruplex formation when a G-quadruplex-forming sequence is partially fed into the T7 RNAP to a position about seven nucleotides away from the front of RNA synthesis. Based on these facts, we propose a model for the strand-biased formation of G-quadruplexes in transcribed DNA duplexes. PMID:26074352

  1. Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene.

    PubMed

    Konishi, Yuki; Hayashi, Hiroshi; Suzuki, Hiromu; Yamamoto, Eiichiro; Sugisaki, Hajime; Higashimoto, Hiroko

    2015-09-01

    Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation-bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight-by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R(2)=0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R(2)=0.864 and R(2)=0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison. PMID:25979762

  2. Recombinant selection by microinjection: a simple cDNA cloning procedure for production of exclusively sense RNA transcripts.

    PubMed

    Digweed, M; Günthert, U

    1989-11-15

    A new strategy for cDNA cloning is presented, designed particularly for identification of recombinants by functional analysis, after microinjection into somatic cells. First-strand synthesis is primed by the oligodeoxyribonucleotide: (formula; see text) After second-strand synthesis and blunting, double-stranded cDNA is formed, which carries restriction sites for NotI and ApaI downstream from the coding sequence. The cDNA is ligated into a plasmid, between two promoters for phage T7 and T3 RNA polymerases. Following transfection and amplification in Escherichia coli, plasmids are extracted from the library or sublibraries. Linearisation with NotI, prior to in vitro transcription, cleaves the plasmids between the 3'-end of the coding sequence and the adjacent promoter and thus ensures that only sense RNAs, suitable for microinjection, are produced after addition of the RNA polymerases. Use of NotI, a rare cutter in the human genome, should ensure that the cDNA inserts are not damaged during linearisation. In the unlikely event that this does happen, a site for ApaI is also available. The method is demonstrated for the human adenine phosphoribosyltransferase-encoding gene. PMID:2591743

  3. Oncolytic adenovirus-expressed RNA interference of O6-methylguanine DNA methyltransferase activity may enhance the antitumor effects of temozolomide

    PubMed Central

    CHEN, XIN-JUN; ZHANG, KAI; XIN, YONG; JIANG, GUAN

    2014-01-01

    Temozolomide (TMZ) is an example of an alkylating agent, which are known to be effective anticancer drugs for the treatment of various solid tumors, including glioma and melanoma. TMZ acts predominantly through the mutagenic product O6-methylguanine, a cytotoxic DNA lesion. The DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which functions in the resistance of cancers to TMZ, can repair this damage. RNA interference (RNAi) has been previously shown to be a potent tool for the knockdown of genes, and has potential for use in cancer treatment. Oncolytic adenoviruses not only have the ability to destroy cancer cells, but may also be possible vectors for the expression of therapeutic genes. We therefore hypothesized that the oncolytic virus-mediated RNAi of MGMT activity may enhance the antitumor effect of TMZ and provide a promising method for cancer therapy. PMID:25295108

  4. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient. PMID:23870991

  5. NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation

    SciTech Connect

    He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga; Zhu, Jianhua; Lu, Jian; Bressan, Ray A.; Pikaard, Craig S.; Wang, Co-Shine; Zhu, Jian-Kang

    2009-01-01

    RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

  6. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    PubMed

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. PMID:25950935

  7. Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats

    PubMed Central

    Hu, Shuanggang; Zhang, Jinsong; Xie, Shengsong; Ma, Wubin; Ni, Minjie; Tang, Chunhua; Zhou, Lu; Zhou, Yuchuan; Liu, Mofang; Li, Yixue; Zhang, Yonglian

    2015-01-01

    Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in sperm maturation / storage of caput / cauda epididymis. PMID:25901964

  8. Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats.

    PubMed

    Chu, Chen; Zheng, Guangyong; Hu, Shuanggang; Zhang, Jinsong; Xie, Shengsong; Ma, Wubin; Ni, Minjie; Tang, Chunhua; Zhou, Lu; Zhou, Yuchuan; Liu, Mofang; Li, Yixue; Zhang, Yonglian

    2015-01-01

    Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97 kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in sperm maturation / storage of caput / cauda epididymis. PMID:25901964

  9. New Dark Matter Detectors using DNA or RNA for Nanometer Tracking

    E-print Network

    Andrzej Drukier; Katherine Freese; Alejandro Lopez; David Spergel; Charles Cantor; George Church; Takeshi Sano

    2015-01-11

    Weakly Interacting Massive Particles (WIMPs) may constitute most of the matter in the Universe. The ability to detect the directionality of recoil nuclei will considerably facilitate detection of WIMPs. In this paper we propose a novel type of dark matter detector: detectors made of DNA or RNA could provide nanometer resolution for tracking, an energy threshold of 0.5 keV, and can operate at room temperature. When a WIMP from the Galactic Halo elastically scatters off of a nucleus in the detector, the recoiling nucleus then traverses hundreds of strings of single stranded nucleic acids (ssNA) with known base sequences and severs ssNA strands along its trajectory. The location of the break can be identified by amplifying and identifying the segments of cut ssNA using techniques well known to biologists. Thus the path of the recoiling nucleus can be tracked to nanometer accuracy. In one such detector concept, the transducers are nanometer-thick Au-foils of 1m x 1m, and the direction of recoiling nuclei is measured by "NA Tracking Chamber" consisting of ordered array of ssNA strands. Polymerase Chain Reaction (PCR) and ssNA sequencing are used to read-out the detector. The proposed detector is smaller and cheaper than other alternatives: 1 kg of gold and 0.1 to 4 kg of ssNA (depending on length and strand density), packed into 0.01m$^3$, can be used to study 10 GeV WIMPs. A variety of other detector target elements could be used in this detector to optimize for different WIMP masses and to identify WIMP properties. By leveraging advances in molecular biology, we aim to achieve about 1,000-fold better spatial resolution than in conventional WIMP detectors at reasonable cost.

  10. DNA DNA DNA (d)DNA DNA DNA

    E-print Network

    Hagiya, Masami

    DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

  11. A Tudor staphylococcal nuclease from Penaeus monodon: cDNA cloning and its involvement in RNA interference.

    PubMed

    Phetrungnapha, Amnat; Panyim, Sakol; Ongvarrasopone, Chalermporn

    2011-09-01

    RNA interference (RNAi) plays an important role in an antiviral defense in shrimp. RNAi technology has been extensively used for inhibition of viral replication and studying gene function. However, the mechanism of shrimp RNAi pathway is still poorly understood. In this study, we identified and characterized an additional protein in the RNAi pathway, Tudor staphylococcal nuclease from Penaeus monodon (PmTSN). The full-length cDNA of PmTSN is 2897 bp, with an open reading frame encoding a putative protein of 889 amino acids. Phylogenetic analysis and domain structure comparison revealed that PmTSN is more closely related to vertebrate TSN by sharing the amino acid sequence identity of 57% with TSN of zebrafish. This represents a new type of TSN proteins by exhibiting the four tandem repeat of staphylococcal nuclease-like domain (SN), followed by a Tudor and a partially truncated C-terminal SN domain. Knockdown of PmTSN by dsRNA targeting SN3 domain resulted in the impairment of dsRNA targeting PmRab7 gene to silence PmRab7 expression. In addition, the efficiency of dsRNA targeting YHV-protease gene inhibiting yellow head virus replication was decreased in the PmTSN-knockdown shrimps. Our results imply that PmTSN is involved in dsRNA-mediated gene silencing in shrimp and thus we identified the additional protein involved in shrimp RNAi pathway. PMID:21745576

  12. Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine

    Microsoft Academic Search

    V Schirrmacher; P Förg; W Dalemans; K Chlichlia; Y Zeng; P Fournier; P von Hoegen

    2000-01-01

    To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for ?-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination

  13. A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus

    Microsoft Academic Search

    Guang Yang; Munir Mawassi; Lilach Ashoulin; Ron Gafny; Victor Gaba; Amit Gal-On; Moshe Bar-Joseph

    A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza clostero- virus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5« and 3« ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of

  14. Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

    Microsoft Academic Search

    Chuanwu Xi; Michal Balberg; Stephen A. Boppart; Lutgarde Raskin

    2003-01-01

    DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA

  15. Abnormal expression and mutation of p53 in cervical cancer--a study at protein, RNA and DNA levels.

    PubMed Central

    Ngan, H Y; Tsao, S W; Liu, S S; Stanley, M

    1997-01-01

    OBJECTIVES: The objectives of this study are to document the status of p53 expression and mutation in cervical cancer at protein, RNA and DNA levels and to relate this to the presence of HPV. MATERIALS AND METHODS: Biopsy specimens from one hundred and three squamous cell carcinoma of the cervix and histologically normal ectocervix were analysed. Fresh tissues were extracted for protein, RNA and DNA and flash frozen tissue cryostat sectioned for immunohistochemical staining. HPV DNA status was determined by PCR using L1 consensus primers and typed for HPV 16 and 18 with E6 specific primers. p53 expression was determined at the protein level by Western blotting on protein extracts and at RNA level by Northern blotting. RESULTS: There was no p53 overexpression or mutation detectable in the protein extracts. Three of 65 (4.6%) of the carcinomas were positive for p53 by immunostaining with the polyclonal antibody CM1. Overexpression at the RNA level was detected in 2 of 32 (6.3%) carcinomas. p53 mutation was screened for by PCR/SSCP (single strand conformation polymorphism) followed by sequencing to define the site of mutation. Two of the cervical cancers (2.0%) showed mutation in p53 in exons 7 or 8. The mutation rate in HPV positive tumours was 1.2% (1/81) and in HPV negative tumours was 5.2% (1/19). CONCLUSION: p53 overexpression or mutation does not seem to play a significant role in cervical carcinomas. Images PMID:9155557

  16. Evaluation of the DNA-dependent RNA polymerase ?-subunit gene (rpoB) for phytoplasma classification and phylogeny.

    PubMed

    Valiunas, Deividas; Jomantiene, Rasa; Davis, Robert Edward

    2013-10-01

    Phytoplasmas are classified into 16Sr groups and subgroups and 'Candidatus Phytoplasma' species, largely or entirely based on analysis of 16S rRNA gene sequences. Yet, distinctions among closely related 'Ca. Phytoplasma' species and strains based on 16S rRNA genes alone have limitations imposed by the high degree of rRNA nucleotide sequence conservation across diverse phytoplasma lineages and by the presence in a phytoplasma genome of two, sometimes sequence-heterogeneous, copies of the 16S rRNA gene. Since the DNA-dependent RNA polymerase (DpRp) ?-subunit gene (rpoB) exists as a single copy in the phytoplasma genome, we explored the use of rpoB for phytoplasma classification and phylogenetic analysis. We sequenced a clover phyllody (CPh) phytoplasma genetic locus containing ribosomal protein genes, a complete rpoB gene and a partial rpoC gene encoding the ?'-subunit of DpRp. Primers and reaction conditions were designed for PCR-mediated amplification of rpoB gene fragments from diverse phytoplasmas. The rpoB gene sequences from phytoplasmas classified in groups 16SrI, 16SrII, 16SrIII, 16SrX and 16SrXII were subjected to sequence similarity and phylogenetic analyses. The rpoB gene sequences were more variable than 16S rRNA gene sequences, more clearly distinguishing among phytoplasma lineages. Phylogenetic trees based on 16S rRNA and rpoB gene sequences had similar topologies, and branch lengths in the rpoB tree facilitated distinctions among closely related phytoplasmas. Virtual RFLP analysis of rpoB gene sequences also improved distinctions among closely related lineages. The results indicate that the rpoB gene provides a useful additional marker for phytoplasma classification that should facilitate studies of disease aetiology and epidemiology. PMID:24097832

  17. Unique nucleocytoplasmic dsDNA and +ssRNA viruses are associated with the dinoflagellate endosymbionts of corals

    PubMed Central

    Correa, Adrienne M S; Welsh, Rory M; Vega Thurber, Rebecca L

    2013-01-01

    The residence of dinoflagellate algae (genus: Symbiodinium) within scleractinian corals is critical to the construction and persistence of tropical reefs. In recent decades, however, acute and chronic environmental stressors have frequently destabilized this symbiosis, ultimately leading to coral mortality and reef decline. Viral infection has been suggested as a trigger of coral–Symbiodinium dissociation; knowledge of the diversity and hosts of coral-associated viruses is critical to evaluating this hypothesis. Here, we present the first genomic evidence of viruses associated with Symbiodinium, based on the presence of transcribed +ss (single-stranded) RNA and ds (double-stranded) DNA virus-like genes in complementary DNA viromes of the coral Montastraea cavernosa and expressed sequence tag (EST) libraries generated from Symbiodinium cultures. The M. cavernosa viromes contained divergent viral sequences similar to the major capsid protein of the dinoflagellate-infecting +ssRNA Heterocapsa circularisquama virus, suggesting a highly novel dinornavirus could infect Symbiodinium. Further, similarities to dsDNA viruses dominated (?69%) eukaryotic viral similarities in the M. cavernosa viromes. Transcripts highly similar to eukaryotic algae-infecting phycodnaviruses were identified in the viromes, and homologs to these sequences were found in two independently generated Symbiodinium EST libraries. Phylogenetic reconstructions substantiate that these transcripts are undescribed and distinct members of the nucleocytoplasmic large DNA virus (NCLDVs) group. Based on a preponderance of evidence, we infer that the novel NCLDVs and RNA virus described here are associated with the algal endosymbionts of corals. If such viruses disrupt Symbiodinium, they are likely to impact the flexibility and/or stability of coral–algal symbioses, and thus long-term reef health and resilience. PMID:22791238

  18. The Arabidopsis RNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci[W][OA

    PubMed Central

    Havecker, Ericka R.; Wallbridge, Laura M.; Hardcastle, Thomas J.; Bush, Maxwell S.; Kelly, Krystyna A.; Dunn, Ruth M.; Schwach, Frank; Doonan, John H.; Baulcombe, David C.

    2010-01-01

    Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5? adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5? nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications. PMID:20173091

  19. A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Telomerase in Response to DNA Damage.

    PubMed

    Xu, Hengyi; Nelson, Andrew D L; Shippen, Dorothy E

    2015-06-01

    Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3' terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2?) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault. PMID:26075395

  20. A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Telomerase in Response to DNA Damage

    PubMed Central

    Xu, Hengyi; Nelson, Andrew D. L.; Shippen, Dorothy E.

    2015-01-01

    Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3’ terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2?) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault. PMID:26075395

  1. Three RNA cells for ribosomal lineages and three DNA viruses to replicate their genomes: a hypothesis for the origin of cellular domain.

    PubMed

    Forterre, Patrick

    2006-03-01

    The division of the living world into three cellular domains, Archaea, Bacteria, and Eukarya, is now generally accepted. However, there is no consensus about the evolutionary relationships among these domains, because all of the proposed models have a number of more or less severe pitfalls. Another drawback of current models for the universal tree of life is the exclusion of viruses, otherwise a major component of the biosphere. Recently, it was suggested that the transition from RNA to DNA genomes occurred in the viral world, and that cellular DNA and its replication machineries originated via transfers from DNA viruses to RNA cells. Here, I explore the possibility that three such independent transfers were at the origin of Archaea, Bacteria, and Eukarya, respectively. The reduction of evolutionary rates following the transition from RNA to DNA genomes would have stabilized the three canonical versions of proteins involved in translation, whereas the existence of three different founder DNA viruses explains why each domain has its specific DNA replication apparatus. In that model, plasmids can be viewed as transitional forms between DNA viruses and cellular chromosomes, and the formation of different levels of cellular organization (prokaryote or eukaryote) could be traced back to the nature of the founder DNA viruses and RNA cells. PMID:16505372

  2. Binding of the Termination Factor Nsi1 to Its Cognate DNA Site Is Sufficient To Terminate RNA Polymerase I Transcription In Vitro and To Induce Termination In Vivo

    PubMed Central

    Merkl, Philipp; Perez-Fernandez, Jorge; Pilsl, Michael; Reiter, Alarich; Williams, Lydia; Gerber, Jochen; Böhm, Maria; Deutzmann, Rainer; Griesenbeck, Joachim

    2014-01-01

    Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3? end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination. PMID:25092870

  3. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

    PubMed

    Vispé, Stéphane; Deroide, Arthur; Davoine, Emeline; Desjobert, Cécile; Lestienne, Fabrice; Fournier, Lucie; Novosad, Natacha; Bréand, Sophie; Besse, Jérôme; Busato, Florence; Tost, Jörg; De Vries, Luc; Cussac, Didier; Riond, Joëlle; Arimondo, Paola B

    2015-06-20

    5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia. PMID:25948775

  4. Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae.

    PubMed Central

    Hookey, J V; Birtles, R J; Saunders, N A

    1995-01-01

    A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species within the family Legionellaceae. The sizes of the PCR products varied from 1,353 to 350 bp. Strains of Legionella pneumophila were characterized as having products of approximately 900 and 530 bp, and L. birminghamensis had products of 1,390, 960, and 380 bp. Of the 38 species of legionellae examined, only 7 were indistinguishable (L. erythra from L. rubrilucens, L. anisa or L. cherrii from L. tucsonensis, and L. quateirensis from L. shakespearei). Two environmental isolates were identified as L. pneumophila. Strain LLAP-3, which was a symbiont of amoebae, could not be associated with any Legionella sp. studied. PMID:7494031

  5. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon).

    PubMed

    Unajak, Sasimanas; Boonsaeng, Vichai; Jitrapakdee, Sarawut

    2006-10-01

    We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection. PMID:16938476

  6. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown.

    PubMed

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai; Helleday, Thomas; Muschel, Ruth J; Taylor, Steve; Yoshimura, Michio; Hickson, Ian D; Bernhard, Eric J; McKenna, W Gillies

    2010-04-01

    The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization. PMID:20233878

  7. RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

    PubMed

    Haas, C; Hanson, E; Anjos, M J; Ballantyne, K N; Banemann, R; Bhoelai, B; Borges, E; Carvalho, M; Courts, C; De Cock, G; Drobnic, K; Dötsch, M; Fleming, R; Franchi, C; Gomes, I; Hadzic, G; Harbison, S A; Harteveld, J; Hjort, B; Hollard, C; Hoff-Olsen, P; Hüls, C; Keyser, C; Maroñas, O; McCallum, N; Moore, D; Morling, N; Niederstätter, H; Noël, F; Parson, W; Phillips, C; Popielarz, C; Roeder, A D; Salvaderi, L; Sauer, E; Schneider, P M; Shanthan, G; Court, D Syndercombe; Turanská, M; van Oorschot, R A H; Vennemann, M; Vidaki, A; Zatkalíková, L; Ballantyne, J

    2014-01-01

    The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology. PMID:24315610

  8. Mitochondrial DNA of Chloramphenicol-Resistant Mouse Cells Contains a Single Nucleotide Change in the Region Encoding the 3' End of the Large Ribosomal RNA

    Microsoft Academic Search

    Hugues Blanc; Catharine T. Wright; Maureen J. Bibb; Douglas C. Wallace; David A. Clayton

    1981-01-01

    The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction

  9. The fate and role of macromolecules synthesized during mammalian oocyte meiotic maturation.

    E-print Network

    Paris-Sud XI, Université de

    The fate and role of macromolecules synthesized during mammalian oocyte meiotic maturation. I during meiotic maturation. Tritiated uridine and lysine or 35S-methio- nine were introduced maturation it was found that an intensive RNA synthesis site appeared in asso- ciation with condensing

  10. MACROMOLECULES FACILITATE THE TRANSPORT OF TRACE ORGANICS

    EPA Science Inventory

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

  11. Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication.

    PubMed

    Zhu, Bin; Lee, Seung-Joo; Tan, Min; Wang, En-Duo; Richardson, Charles C

    2012-05-22

    DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3'-terminal sequence (5'-ACCA-3') of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition. PMID:22566619

  12. Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

    PubMed Central

    Horton, J K; Srivastava, D K; Zmudzka, B Z; Wilson, S H

    1995-01-01

    Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-pol is down-regulated in the antisense cell line. Such a sensitization is anticipated only where beta-pol is rate-limiting in a DNA repair pathway. A number of agents were tested: cis-diamminedichloroplatinum II (cisplatin); 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU); ionizing radiation and the radio-mimetic drug bleomycin; the bifunctional alkylating agents nitrogen mustard and L-phenylalanine mustard (melphalan); the monofunctional alkylating agent methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. In the cases of cisplatin and UV radiation, a significant enhancement of cytotoxicity was observed. Damage as a result of both of these agents is thought to be repaired by the nucleotide excision repair (NER) pathway. The results suggest that, in this cell line, beta-pol is involved in and is rate-limiting in NER. We propose that down-regulation of beta-pol by antisense approaches might be used to enhance the cytotoxic effects of cisplatin and other DNA damaging chemotherapeutic agents. Images PMID:7479021

  13. Dynamic separation of macromolecules under temperature gradient

    NASA Astrophysics Data System (ADS)

    Maeda, Yusuke; Buguin, Axel; Libchaber, Albert

    2011-03-01

    Thermophoresis is a motion of suspensions in a fluid that are subjected to a temperature gradient. Although its effect is widely studied in case of single solute in water, little is known about how the mixture of different solutes is affected. We heated water with an infrared laser by ?Tmax = 5C and ? T = 0.25C/um to induce thermophoresis of polyethylene glycol (PEG) and DNA. PEG is depleted from the hot region and results in a stationary gradient of its high volume fraction ? . Under this high concentration of PEG, DNA of small concentration is submitted to thermophoresis and osmotic pressure difference. The DNA shows regime of depletion, ring-like localization and accumulation as the volume fraction of PEG increases. As the osmotic force depends on the size of trapped solutes, DNA of different size accumulates at different regions. Depending whether the DNA size is below or above 5kbp a different scaling of position versus DNA size is observed. Thermal separation is a general phenomenon. It applies also to RNA and microbeads. YTM is supported by JSPS fellowship and M.Josee-H.Kravis fellowship from the Rockefeller University.

  14. Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference.

    PubMed

    Dechklar, Manasave; Udomkit, Apinunt; Panyim, Sakol

    2008-03-21

    RNA interference (RNAi) has recently become a promising strategy for therapeutic of several viral diseases including those in the black tiger shrimp Penaeus monodon. However, the protein components that play role in RNAi in P. monodon have not yet been identified. Here, we report the cloning and functional characterization of a cDNA encoding Argonaute, a principal constituent of RNAi pathway in P. monodon. P. monodon's Argonaute (Pem-AGO) exhibited the two signature domains, PAZ and PIWI. Substantial level of Pem-ago expression could be suppressed by double-stranded RNA (dsRNA) that targeted PAZ coding sequence in shrimp primary culture of Oka cells. The Pem-ago depleted cells showed impaired RNAi as the expression of an endogenous gene was rescued from the dsRNA-mediated silencing in these cells. Our results imply that Pem-ago is required for effective RNAi in P. monodon and thus identify the first protein constituent of RNAi machinery in penaeid shrimp. PMID:18201549

  15. Small RNA- and DNA-based gene therapy for the treatment of liver cirrhosis, where we are?

    PubMed Central

    Kim, Kyung-Hyun; Park, Kwan-Kyu

    2014-01-01

    Chronic liver diseases with different aetiologies rely on the chronic activation of liver injuries which result in a fibrogenesis progression to the end stage of cirrhosis and liver failure. Based on the underlying cellular and molecular mechanisms of a liver fibrosis, there has been proposed several kinds of approaches for the treatment of liver fibrosis. Recently, liver gene therapy has been developed as an alternative way to liver transplantation, which is the only effective therapy for chronic liver diseases. The activation of hepatic stellate cells, a subsequent release of inflammatory cytokines and an accumulation of extracellular matrix during the liver fibrogenesis are the major obstacles to the treatment of liver fibrosis. Several targeted strategies have been developed, such as antisense oligodeoxynucleotides, RNA interference and decoy oligodeoxynucleotides to overcome this barriers. With this report an overview will be provided of targeted strategies for the treatment of liver cirrhosis, and particularly, of the targeted gene therapy using short RNA and DNA segments. PMID:25356032

  16. Posttranscriptional Self-Regulation by the Lyme Disease Bacterium's BpuR DNA/RNA-Binding Protein

    PubMed Central

    Jones, Grant S.; Verma, Ashutosh; Brown, Nicholas A.; Antonicello, Alyssa D.; Chenail, Alicia M.

    2013-01-01

    Bacteria require explicit control over their proteomes in order to compete and survive in dynamic environments. The Lyme disease spirochete Borrelia burgdorferi undergoes substantial protein profile changes during its cycling between vector ticks and vertebrate hosts. In an effort to understand regulation of these transitions, we recently isolated and functionally characterized the borrelial nucleic acid-binding protein BpuR, a PUR domain-containing protein. We now report that this regulatory protein governs its own synthesis through direct interactions with bpuR mRNA. In vitro and in vivo techniques indicate that BpuR binds with high affinity and specificity to the 5? region of its message, thereby inhibiting translation. This negative feedback could permit the bacteria to fine-tune cellular BpuR concentrations. These data add to the understanding of this newly described class of prokaryotic DNA- and RNA-binding regulatory proteins. PMID:23974034

  17. Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges

    PubMed Central

    Naiser, Thomas; Ehler, Oliver; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

    2008-01-01

    Background The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. Results We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form). Conclusion We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. PMID:18477387

  18. Fathead minnow vitellogenin: Complementary DNA sequence and messenger RNA and protein expression after 17{beta}-estradiol treatment

    SciTech Connect

    Korte, J.J.; Kahl, M.D.; Jensen, K.M.; Pasha, M.S.; Parks, L.G.; LeBlanc, G.A.; Ankley, G.T.

    2000-04-01

    Induction of vitellogenin (VTG) in oviparous animals has been proposed as a sensitive indicator of environmental contaminants that activate the estrogen receptor. In the present study, a sensitive ribonuclease protection assay (RPA) for VTG messenger RNA (mRNA) was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening. The utility of this method was compared with an enzyme-linked immunosorbent assay (ELISA) specific for fathead minnow VTG protein. Assessment of the two methods included kinetic characterization of the plasma VTG protein and hepatic VTG mRNA levels in male fathead minnows following intraperitoneal injections of 17{beta}-estradiol (E2) at two dose levels (0.5, 5.0 mg/kg). Initial plasma E2 concentrations were elevated in a dose-dependent manner but returned to normal levels within 2 d. Lover VTG mRNA was detected within 4 h, reached a maximum around 48 h, and returned to normal levels in about 6 d. Plasma VTG protein was detectable within 16 h of treatment reached maximum levels at about 72 h. and remained near these maximum levels for at least 18 d. While the RPA was about 1,000 times more sensitive than the ELISA, the ELISA appears superior for routine screening tests. The ELISA method is relatively simple to perform and, because males lack a clearance mechanism for VTG, the protein remains at relatively high concentrations in the plasma for an extended period of time. As part of the development of the RPA, the complementary DNA (cDNA) sequence for fathead minnow VTG was determined and the deduced amino acid sequence compared with VTG sequences for other fish species.

  19. YB-1 and MTA1 protein levels and not DNA or mRNA alterations predict for prostate cancer recurrence

    PubMed Central

    Sheridan, Christine Moore; Grogan, Tristan R.; Nguyen, Hao G.; Galet, Colette; Rettig, Matthew B.; Hsieh, Andrew C.; Ruggero, Davide

    2015-01-01

    Attempts to identify biomarkers to detect prostate tumorigenesis, and thus minimize prostate cancer progression and inform treatment decisions have primarily focused on alterations at the DNA and mRNA levels, ignoring alterations at the level of protein synthesis control. We have previously shown that the PI3K-AKT-mTOR pathway, frequently deregulated in prostate cancer, specifically induces the synthesis of proteins that contribute to metastasis, most notably YB-1 and MTA1, without altering mRNA levels thereby demonstrating the importance of translation control in driving the expression of these genes in cancer. Here, we analyze genomic sequencing and mRNA expression databases, as well as protein expression employing an annotated tissue microarray generated from 332 prostate cancer patients with 15 years of clinical follow-up to determine the combined prognostic capability of YB-1 and MTA1 alterations in forecasting prostate cancer outcomes. Remarkably, protein abundance, but not genomic or transcriptional alterations of YB-1 and MTA1, is predictive of disease recurrence, exhibiting a dose-dependent effect on time to PSA recurrence, an indicator of tumor relapse. Moreover, high protein levels of YB-1 and MTA1 are associated with a 3-fold increased risk for requiring future hormone therapy or radiation therapy. Importantly, YB-1 and MTA1 protein levels significantly increase the predictive capacity of a clinical model for prostate cancer recurrence. These findings demonstrate that protein abundance of YB-1 and MTA1, irrespective of DNA or mRNA status, can predict for prostate cancer relapse and uncover a vast underappreciated repository of biomarkers regulated at the level of protein expression. PMID:25797255

  20. Bioorganometallic chemistry: synthesis, structure, and molecular recognition chemistry of (? 5-pentamethylcyclopentadienyl)rhodium–DNA\\/RNA complexes in water

    Microsoft Academic Search

    Richard H. Fish

    1999-01-01

    A review of the aqueous bioorganometallic chemisty of DNA\\/RNA nucleobases and of the co-factor, nicotinamide adenine dinucleotide, with an (?5-pentamethylcyclopentadienyl)rhodium aqua complex, [Cp*Rh(H2O)3](OTf)2, at various pH values, will be presented. The unique structures of the Cp*Rh complexes with adenine, guanine, cytosine, thymine, and nicotinamide adenine dinucleotide bioligands were determined by a combination of 1H- and 31P-NMR, ESI\\/MS, and single crystal

  1. Evidence for RNA linked to nascent DNA in HeLa cells

    Microsoft Academic Search

    DOMINIQUE DES GOUTTES OLGIATI; BEATRIZ G. T. POGO; SAMUEL DALES

    1976-01-01

    Rapidly labeled, i.e., nascent, DNA from HeLa cells was separated from the bulk DNA by ultracentrifugat ion. Further characterization of the rapidly labeled component revealed that its sedimentation coefficient is in the range of 4S and that it exists in a single- and double-stranded conformation. Moreover, analysis by nitrocellulose chromatography and CsSO4 density sedimentation of the nascent DNA labeled with

  2. Submillimeter wave spectroscopy of biological macromolecules

    NASA Astrophysics Data System (ADS)

    Globus, Tatiana

    2005-03-01

    The recently emergence of submillimeter-wave or terahertz (THz) spectroscopy of biological molecules has demonstrated the capability to detect low-frequency internal molecular vibrations involving the weakest hydrogen bonds of the DNA base pairs and/or non-bonded interactions. These multiple bonds, although having only ˜ 5% of the strength of covalent bonds, stabilize the structure of bio-polymers, by holding the two strands of the DNA double helix together, or polypeptides together in different secondary structure conformations. There will be a review of THz-frequency transmission (absorption) results for biological materials obtained from Fourier Transform Infrared (FTIR) spectroscopy during the last few years^1,2. Multiple resonances, due to low frequency vibrational modes within biological macromolecules, have been unambiguously demonstrated in qualitative agreement with theoretical prediction, thereby confirming the fundamental physical nature of observed resonance features. The discovery of resonance character of interaction between THz radiation and biological materials opens many possible applications for THz spectroscopy technique in biological sensing and biomedicine using multiple resonances as distinctive spectral fingerprints. However, many issues still require investigation. Kinetics of interactions with radiation at THz has not been studied and vibrational lifetimes have not been measured directly as a function of frequency. The strength of resonant modes of bio-molecules in aqueous environment and strong dependence of spectra on molecular orientation need explanation. Vibrational modes have not been assigned to specific motions within molecules. THz spectroscopy of bio-polymers makes it only in first steps. 1. T. Globus, D. Woolard, M. Bykhovskaia, B. Gelmont, L. Werbos, A. Samuels. International Journal of High Speed Electronics and Systems (IJHSES), 13, No. 4, 903-936 (2003). 2. T. Globus, T. Khromova, D. Woolard and B. Gelmont. Proceedings of SPIE Vol. 5268-2, 10-18 (2004)

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

    Microsoft Academic Search

    Lakshminarayan M Iyer; Saraswathi Abhiman; L Aravind

    2008-01-01

    : Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both

  4. Synthesis and DNA/RNA Binding Properties of Conformationally Constrained Pyrrolidinyl PNA with a Tetrahydrofuran Backbone Deriving from Deoxyribose.

    PubMed

    Sriwarom, Pitchanun; Padungros, Panuwat; Vilaivan, Tirayut

    2015-07-17

    Sugar-derived cyclic ?-amino acids are important building blocks for designing of foldamers and other biomimetic structures. We report herein the first synthesis of a C-activated N-Fmoc-protected trans-(2S,3S)-3-aminotetrahydrofuran-2-carboxylic acid as a building block for Fmoc solid phase peptide synthesis. Starting from 2-deoxy-d-ribose, the product is obtained in a 6.7% overall yield following an 11-step reaction sequence. The tetrahydrofuran amino acid is used as a building block for a new peptide nucleic acid (PNA), which exhibits excellent DNA binding affinity with high specificity. It also shows preference for binding to DNA over RNA and specifically in the antiparallel orientation. In addition, the presence of the hydrophilic tetrahydrofuran ring in the PNA structure reduces nonspecific interactions and self-aggregation, which is a common problem in PNA due to its hydrophobic nature. PMID:26083668

  5. Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis.

    PubMed

    Cui, X M; Lo, C K; Yip, K L; Dong, T T; Tsim, K W

    2003-06-01

    The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species. PMID:12865989

  6. Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum).

    PubMed

    May, C E; Appels, R

    1987-09-01

    Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed. PMID:24240218

  7. Nucleic acid concentrations (DNA, RNA) in the continental and deep-sea sediments of the eastern Mediterranean: relationships with seasonally varying organic inputs and bacterial dynamics

    NASA Astrophysics Data System (ADS)

    Danovaro, R.; Dell'anno, A.; Pusceddu, A.; Fabiano, M.

    1999-06-01

    In order to study temporal variations of the genetic material in the continental shelf and deep-sea sediments of the extremely oligotrophic Cretan Sea, samples were collected on seasonal basis from August 1994 to September 1995, with a multiple corer, at seven stations (from 40 to 1540 m depth). Surface sediments (0-1 cm) were sub-sampled and analyzed for nucleic acid content (DNA, RNA) and bacterial density. DNA concentrations in the sediments were high (on annual average, 25.0 ?g g -1) and declined with increasing water depth, ranging from 3.5 to 55.2 ?g g -1. DNA concentrations displayed wide temporal changes also at bathyal depths confirming the recent view of the large variability of the deep-sea environments. Also RNA concentrations decreased with increasing water depth (range: 0.4-29.9 ?g g -1). The ratio of RNA to DNA did not show a clear spatial pattern but was characterized by significant changes between sampling periods. DNA concentrations were significantly correlated with protein and phytopigment concentrations in the sediment, indicating a possible relationship with the inputs of primary organic matter from the photic layer. Bacterial densities were generally high (range: 0.9-4.6×10 8 cells g -1) compared to other deep-sea environments and decreased with increasing water depth. Estimates of the bacterial contribution to the sedimentary genetic material indicated that bacterial-DNA accounted, on annual average, for a small fraction of the total DNA pool (4.3%) but that bacterial-RNA represented a significant fraction of the total sedimentary RNA (26%). Bacterial contribution to nucleic acids increased, even though irregularly, with increasing depth. In deep-sea sediments, changes in RNA concentrations appear to be largely dependent upon bacterial dynamics. Estimates of the overall living contribution to the DNA pools (i.e. microbial plus meiofaunal DNA) indicated that the large majority (about 90%) of the DNA in continental and deep-sea sediments of the eastern Mediterranean was detrital. The non-living DNA pools reach extremely high concentrations up to 0.41 g DNA m -2 cm -1. Thus, especially in deep benthic habitats, characterized by low inputs of labile organic compounds, detrital DNA could represent a suitable and high quality food source or a significant reservoir of nucleic acid precursors for benthic metabolism.

  8. Ribonuclease-resistant RNA Controls (Armored RNA) for Reverse Transcription-PCR, Branched DNA, and Genotyping Assays for Hepatitis C Virus

    Microsoft Academic Search

    Cindy R. WalkerPeach; Matthew Winkler; Dwight B. DuBois; Brittan L. Pasloske

    Background: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA® technology is a means for producing RNA that is com- pletely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA

  9. A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.

    PubMed

    Yang, G; Mawassi, M; Ashoulin, L; Gafny, R; Gaba, V; Gal-On, A; Bar-Joseph, M

    1997-07-01

    A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA. PMID:9225053

  10. Helicobacter pylori RNA polymerase ?-subunit C-terminal domain shows features unique to ?-proteobacteria and binds NikR/DNA complexes

    PubMed Central

    Borin, Brendan N; Tang, Wei; Krezel, Andrzej M

    2014-01-01

    Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the ? subunit of RNA polymerase (?CTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although ?CTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori ?CTD. It is larger than other structurally determined ?CTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among ?-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to ?CTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori ?CTD to NikR:DNA complexes. We also produced ?CTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori ?CTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators. PMID:24442709

  11. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences

    PubMed Central

    Lin, Yanni; Cradick, Thomas J.; Brown, Matthew T.; Deshmukh, Harshavardhan; Ranjan, Piyush; Sarode, Neha; Wile, Brian M.; Vertino, Paula M.; Stewart, Frank J.; Bao, Gang

    2014-01-01

    CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage. PMID:24838573

  12. Proton NMR studies on the covalently linked RNA-DNA hybrid r(GCG)d(TATACGC). Assignment of proton resonances by application of the nuclear Overhauser effect.

    PubMed Central

    Mellema, J R; Haasnoot, C A; van der Marel, G A; Wille, G; van Boeckel, C A; van Boom, J H; Altona, C

    1983-01-01

    Proton NMR spectra of a covalently linked self-complementary RNA X DNA hybrid, r(GCG)-d(TATACGC), are recorded in H2O and D2O. Imino proton resonances as well as the non-exchangeable base and H-1' resonances are unambiguously assigned by means of nuclear. Overhauser effect measurements. Additional information was obtained by 31P NMR and circular dichroism spectra. The RNA parts in the duplex attain full conformational purity and adopt the usual A-RNA conformation. The DNA residues opposite the RNA tract do not adopt an A-type structure completely. Their respective sugar rings still appear to possess a certain conformational freedom. The same holds true for the central d(-TATA-) sequence which forms a DNA X DNA duplex. There appears to be a structural break in this part: the first two residues, T(4) and A(5), are clearly influenced by the adjacent RNA structure, whereas residues T(6) and A(7) behave quite similar to what usually is found in DNA duplexes in aqueous solution. PMID:6193486

  13. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

    PubMed

    Lin, Yanni; Cradick, Thomas J; Brown, Matthew T; Deshmukh, Harshavardhan; Ranjan, Piyush; Sarode, Neha; Wile, Brian M; Vertino, Paula M; Stewart, Frank J; Bao, Gang

    2014-06-01

    CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions ('DNA bulge') or deletions ('RNA bulge') compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage. PMID:24838573

  14. Both OsRecQ1 and OsRDR1 Are Required for the Production of Small RNA in Response to DNA-Damage in Rice

    PubMed Central

    Chen, Hui; Kobayashi, Kappei; Miyao, Akio; Hirochika, Hirohiko; Yamaoka, Naoto; Nishiguchi, Masamichi

    2013-01-01

    Small RNA-mediated gene silencing pathways play important roles in the regulation of development, genome stability and various stress responses in many eukaryotes. Recently, a new type of small interfering RNAs (qiRNAs) approximately 20–21 nucleotides long in Neurospora crassa have been shown to mediate gene silencing in the DNA damage response (DDR) pathway. However, the mechanism for RNA silencing in the DDR pathway is largely unknown in plants. Here, we report that a class of small RNAs (qiRNAs) derived from rDNA was markedly induced after treatment by DNA-damaging agents [ethyl methanesulphonate (EMS and UV-C)], and that aberrant RNAs (aRNAs) as precursors were also highly induced after the DNA damage treatment in rice. However, these RNAs were completely abolished in OsRecQ1 (RecQ DNA helicase homologue) and OsRDR1 (RNA-dependent RNA polymerase homologue) mutant lines where either gene was disrupted by the insertion of rice retrotransposon Tos17 after the same treatment. DNA damage resulted in a more significant increase in cell death and a more severe inhibition of root growth in both mutant lines than in the WT. Together, these results strongly suggest that both OsRecQ1 and OsRDR1 play a pivotal role in the aRNA and qiRNA biogenesis required for the DDR and repair pathway in rice, and it may be a novel mechanism of regulation to the DDR through the production of qiRNA in plants. PMID:23383126

  15. Designs for the manufacture of manipulable plastic DNA/RNA building blocks for learning life science

    E-print Network

    Lemanski, Bethany I

    2013-01-01

    The subject of this thesis is the design of custom injection-molded manipulable DNA building blocks for use in a hands-on life sciences educational kit. The new design of the DNA building blocks is meant to replace the ...

  16. Analysis of bacterial core communities in the central Baltic by comparative RNA–DNA-based fingerprinting provides links to structure–function relationships

    PubMed Central

    Brettar, Ingrid; Christen, Richard; Höfle, Manfred G

    2012-01-01

    Understanding structure–function links of microbial communities is a central theme of microbial ecology since its beginning. To this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central Baltic Sea at four stations, which were up to 450?km apart and at a depth profile representative for the central part (Gotland Deep, 235?m). Bacterial community structure was followed by 16S ribosomal RNA (rRNA)- and 16S rRNA gene-based fingerprints using single-strand conformation polymorphism (SSCP) electrophoresis. Species composition was determined by sequence analysis of SSCP bands. High similarities of the bacterioplankton communities across several hundred kilometers were observed in the surface water using RNA- and DNA-based fingerprints. In these surface communities, the RNA- and DNA-based fingerprints resulted in very different pattern, presumably indicating large difference between the active members of the community as represented by RNA-based fingerprints and the present members represented by the DNA-based fingerprints. This large discrepancy changed gradually over depth, resulting in highly similar RNA- and DNA-based fingerprints in the anoxic part of the water column below 130?m depth. A conceivable mechanism explaining this high similarity could be the reduced oxidative stress in the anoxic zone. The stable communities on the surface and in the anoxic zone indicate the strong influence of the hydrography on the bacterioplankton community structure. Comparative analysis of RNA- and DNA-based community structure provided criteria for the identification of the core community, its key members and their links to biogeochemical functions. PMID:21697960

  17. Analysis of bacterial core communities in the central Baltic by comparative RNA-DNA-based fingerprinting provides links to structure-function relationships.

    PubMed

    Brettar, Ingrid; Christen, Richard; Höfle, Manfred G

    2012-01-01

    Understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. To this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central Baltic Sea at four stations, which were up to 450?km apart and at a depth profile representative for the central part (Gotland Deep, 235?m). Bacterial community structure was followed by 16S ribosomal RNA (rRNA)- and 16S rRNA gene-based fingerprints using single-strand conformation polymorphism (SSCP) electrophoresis. Species composition was determined by sequence analysis of SSCP bands. High similarities of the bacterioplankton communities across several hundred kilometers were observed in the surface water using RNA- and DNA-based fingerprints. In these surface communities, the RNA- and DNA-based fingerprints resulted in very different pattern, presumably indicating large difference between the active members of the community as represented by RNA-based fingerprints and the present members represented by the DNA-based fingerprints. This large discrepancy changed gradually over depth, resulting in highly similar RNA- and DNA-based fingerprints in the anoxic part of the water column below 130?m depth. A conceivable mechanism explaining this high similarity could be the reduced oxidative stress in the anoxic zone. The stable communities on the surface and in the anoxic zone indicate the strong influence of the hydrography on the bacterioplankton community structure. Comparative analysis of RNA- and DNA-based community structure provided criteria for the identification of the core community, its key members and their links to biogeochemical functions. PMID:21697960

  18. 2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

    PubMed

    Sochacka, Elzbieta; Szczepanowski, Roman H; Cypryk, Marek; Sobczak, Milena; Janicka, Magdalena; Kraszewska, Karina; Bartos, Paulina; Chwialkowska, Anna; Nawrot, Barbara

    2015-03-11

    2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon-anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3'-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif. PMID:25690900

  19. Ultrasensitive genotyping with target-specifically generated circular DNA templates and RNA FRET probes.

    PubMed

    Zhou, Haoxian; Wang, Hui; Liu, Chenghui; Wang, Honghong; Duan, Xinrui; Li, Zhengping

    2015-07-25

    A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity. PMID:26095168

  20. Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma.

    PubMed

    Yanase, K; Sugimoto, Y; Tsukahara, S; Oh-Hara, T; Andoh, T; Tsuruo, T

    2000-05-01

    In previous studies, we established two camptothecin (CPT)-resistant sublines, HT-29 / CPT and St-4 / CPT, from the human colon cancer cell line HT-29 and the human stomach cancer cell line St-4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight-fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT-29 / CPT. The longer mRNA (4.0 kb) is the wild-type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58 - 729 or 59 - 730) that caused the in-frame deletion of amino acids 20 - 243 of human topo I. The deleted region is identical to exons 3 - 9 of the TOP1 gene. The expression level of the 3.3-kb mRNA was similar to that of the wild-type mRNA in HT-29 / CPT. St-4 / CPT expressed only the wild-type TOP1 mRNA in lesser amounts than did St-4. Mouse NIH3T3 cells transfected with the wild-type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT-sensitive topo I in the resistant cells. PMID:10835501

  1. Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

    PubMed Central

    Cherepanov, A V; de Vries, S

    2001-01-01

    The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity. PMID:11721015

  2. Structural D/E-rich repeats play multiple roles especially in gene regulation through DNA/RNA mimicry.

    PubMed

    Chou, Chia-Cheng; Wang, Andrew H-J

    2015-07-14

    Aspartic acid and glutamic acid repeats in proteins exhibit strong negative charge distribution and they may play special biological roles. From 39?684 unique structural data in the RCSB Protein Data Bank (PDB), 173 structures were found to contain ordered D/E-rich repeat structures, and 57 of them were related to DNA/RNA functions. The frequency of occurrence of glutamic acid (36.90%) was higher than that of aspartic acid (27.02%). Glycine (2.38%), alanine (2.68%), valine (3.54%), leucine (5.57%), and isoleucine (3.34%), but not methionine (0.91%), were the most abundant hydrophobic residues. The available complex structures suggested that D/E-rich proteins might be involved in DNA mimicry, mRNA processing and regulation of the transcription complex. The region surrounding the D/E-rich repeat sequences plays important roles in the binding specificity toward the target proteins. The numbers and composition of aspartic acid and glutamic acid might also affect binding properties. Aspartic acid and glutamic acid are disorder-promoting residues in the intrinsically disorder proteins. Our findings suggest that the D/E-rich repeats are unique components of intrinsically disordered proteins, which are involved in the gene regulation and could serve as potential druggable fragments or drug targets. PMID:26088262

  3. DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

    PubMed Central

    Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A.; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S.; Lan, Li

    2015-01-01

    Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

  4. In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes.

    PubMed Central

    Berger, C N

    1986-01-01

    A method for in situ hybridization has been developed which detects immunoglobulin-specific mRNA transcripts in single murine B lymphocytes with radiolabelled, immunoglobulin gene-specific single-stranded DNA probes. The method has been applied to myeloma and hybridoma cells and to B lymphocytes at various stages of their maturation from small, resting B cells to Ig-secreting plasma cells. A critical step in the procedure is the treatment of the cells with pronase. The various cell types have been found to be differently susceptible to this treatment. Single-stranded DNA probes of different lengths, i.e., between 26 and 1000 bp, have been employed in the hybridization. The number of silver grains over a cell increases proportionally with the length of the probe and with its concentration in the hybridization reaction. The kinetics of the increase of mu-heavy chain-specific RNA molecules in single cells and the appearance of 'switched', gamma-heavy chain-expressing cells are shown after stimulation of murine B cells with lipopolysaccharide. Images Fig. 1. Fig. 3. PMID:3007120

  5. DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

    PubMed

    Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

    2015-07-01

    Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

  6. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law.

    PubMed

    Rambo, Robert P; Tainer, John A

    2011-08-01

    Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. Here, we consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. We show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay by capturing the information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described here, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, we demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria, as shown here, provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation. PMID:21509745

  7. Association of Poly I:C RNA and Plasmid DNA onto MnO Nanorods Mediated by PAMAM

    PubMed Central

    Parker-Esquivel, Brooke; Flores, Kristin J.; Louiselle, Daniel; Craig, Michael; Dong, Lifeng; Garrad, Richard; Ghosh, Kartik; Wanekaya, Adam; Glaspell, Garry; DeLong, Robert K.

    2012-01-01

    In this study, manganese oxide (MnO) nanorods and its association with polyamidoamine dendrimer (PAMAM) and macromolecular RNA were analyzed. Because manganese is found naturally in cells and tissues and binds proteins and nucleic acids, nanomaterials derived from manganese, such as first generation MnO, may have potential as a biocompatible delivery agent for therapeutic or diagnostic biomedical applications. Nucleic acids have a powerful influence over cell processes, such as gene transcription and RNA processing; however, macromolecular RNA is particularly difficult to stabilize as a nanoparticle and to transport across cell membranes while maintaining structure and function. PAMAM is a cationic, branching dendrimer known to form strong complexes with nucleic acids and to protect them from degradation, and is also considered to be a cell penetrating material. There is currently much interest in polyinosinic:polycytidylic RNA (poly I:C) because of its potent and specific immunogenic properties and as a solo or combination therapy. In order to address this potential, here, as a first step, we used PAMAM to attach poly I:C onto MnO nanorods. Morphology of the MnO nanorods was examined by field emission scanning electron microscopy (FESEM) and their composition by energy dispersive X-ray microanalysis (EDX). Evidence was generated for RNA : PAMAM : MnO nanorod binding by a gel shift assay using gel electrophoresis, a sedimentation assay using UV spectroscopy, and zeta potential shifts using dynamic laser light scattering. The data suggest that RNA was successfully attached to the MnO nanorods using PAMAM, and this suggestion was supported by direct visualization of the ternary complexes with FESEM characterizations. In order to confirm that the associations were biocompatible and taken up by cells, MTT assays were carried out to assess the metabolic activity of HeLa cells after incubation with the complexes and appropriate controls. Subsequently, we performed transfection assays using PAMAM:MnO complexes with pDNA encoding a green fluorescent protein reporter gene instead of RNA. The results suggest that the complexes had minimal impact on metabolic activity, were readily taken up by cells, and the fluorescent protein was expressed. From the evidence, we conclude that complexes of PAMAM:MnO interact with nucleic acids to form associations that are well-tolerated and readily taken up by cells. PMID:22220841

  8. Sequence walkers: a graphical method to display how binding proteins interact with DNA or RNA sequences.

    PubMed Central

    Schneider, T D

    1997-01-01

    A graphical method is presented for displaying how binding proteins and other macromolecules interact with individual bases of nucleotide sequences. Characters representing the sequence are either oriented normally and placed above a line indicating favorable contact, or upside-down and placed below the line indicating unfavorable contact. The positive or negative height of each letter shows the contribution of that base to the average sequence conservation of the binding site, as represented by a sequence logo. These sequence 'walkers' can be stepped along raw sequence data to visually search for binding sites. Many walkers, for the same or different proteins, can be simultaneously placed next to a sequence to create a quantitative map of a complex genetic region. One can alter the sequence to quantitatively engineer binding sites. Database anomalies can be visualized by placing a walker at the recorded positions of a binding molecule and by comparing this to locations found by scanning the nearby sequences. The sequence can also be altered to predict whether a change is a polymorphism or a mutation for the recognizer being modeled. PMID:9336476

  9. ? vs ?-Radical States of One-Electron Oxidized DNA/RNA Bases: A Density Functional Theory Study

    PubMed Central

    Kumar, Anil; Sevilla, Michael D.

    2013-01-01

    As a result of their inherent planarity, DNA base radicals generated by one electron oxidation/reduction or bond cleavage form ?- or ?-radicals. While most DNA base systems form ?-radicals there are a number of nucleobase analogs such as one-electron oxidized 6-azauraci1, 6-azacytosine, and 2-thiothymine or one-electron reduced 5-bromouracil that form more reactive ?-radicals. Elucidating the availability of these states within DNA, base radical electronic structure is important to the understanding of the reactivity of DNA base radicals in different environments. In this work, we address this question by the calculation of the relative energies of ?- and ?-radical states in DNA/RNA bases and their analogs. We used density functional theory B3LYP/6-31++G** method to optimize the geometries of ?- and ?-radicals in Cs symmetry (i.e., planar) in the gas phase and in solution using the polarized continuum model (PCM). The calculations predict that ?- and ?-radical states in one electron oxidized bases of thymine, T(N3-H)•, and uracil, U(N3-H)• are very close in energy, i.e., the ?-radical is only ca. 4 kcal/mol more stable than the ?-radical. For the one electron oxidized radicals of cytosine, C•+, C(N4-H)•, adenine, A•+, A(N6-H)•, and guanine, G•+, G(N2-H)•, G(N1-H)• the ?-radicals are ca. 16 to 41 kcal/mol more stable than their corresponding ?-radicals. Inclusion of solvent (PCM) is found to stabilize the ?- over ?-radical of each of the systems. U(N3-H)• with three discrete water molecules in the gas phase, is found to form a three-electron ? bond between N3 atom of uracil and O atom of a water molecule but on inclusion of full solvation and discrete hydration the ?-radical remains most stable.. PMID:24000793

  10. A G-quadruplex DNA-based, Label-Free and Ultrasensitive Strategy for microRNA Detection

    PubMed Central

    Yan, Li; Yan, Yiyong; Pei, Lei; Wei, Wei; Zhao, Jing

    2014-01-01

    MicroRNAs (miRNAs) have been considered to be potent biomarkers for early disease diagnosis and for cancer therapy. The rapid and selective detection of miRNAs without reverse transcription and labelling is highly desired. Herein, we report a simple and label-free miRNA detection method that is based on the Duplex-Specific Nuclease (DSN)-Assisted simple target miRNA recycling procedure. The interaction of the G-quadruplex DNA structure with N-methyl mesoporphyrin IX (NMM) led to a label-free signal output. Under the optimised conditions, this method allowed for simple, rapid, and sequence-specific detection of miR-141 over a dynamic range from 1 fM to 100?nM with a linear range from 1 pM to 100?nM. Moreover, our method offered an excellent capacity to discriminate between miRNA family members with just one mismatched nucleotide. This simple and label-free strategy holds great potential in applications in biomedical research and in early clinical diagnostics. PMID:25492390

  11. Brickworx builds recurrent RNA and DNA structural motifs into medium- and low-resolution electron-density maps

    PubMed Central

    Chojnowski, Grzegorz; Wale?, Tomasz; Pi?tkowski, Pawe?; Potrzebowski, Wojciech; Bujnicki, Janusz M.

    2015-01-01

    Brickworx is a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs including double-stranded helices. In a first step, the program searches for electron-density peaks that may correspond to phosphate groups; it may also take into account phosphate-group positions provided by the user. Subsequently, comparing the three-dimensional patterns of the P atoms with a database of nucleic acid fragments, it finds the matching positions of the double-stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure is RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real space to find the most likely conformations, including a fit of the sequence to the electron-density map. The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx. PMID:25760616

  12. DNA Nanostructure-based Interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA

    NASA Astrophysics Data System (ADS)

    Wen, Yanli; Pei, Hao; Shen, Ye; Xi, Junjie; Lin, Meihua; Lu, Na; Shen, Xizhong; Li, Jiong; Fan, Chunhai

    2012-11-01

    MicroRNAs (miRNAs) have been identified as promising cancer biomarkers due to their stable presence in serum. As an alternative to PCR-based homogenous assays, surface-based electrochemical biosensors offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and crowding effects at the water-electrode interface. To address such challenges, we herein report a DNA nanostructure-based interfacial engineering approach to enhance binding recognition at the gold electrode surface and drastically improve the detection sensitivity. By employing this novel strategy, we can directly detect as few as attomolar (<1, 000 copies) miRNAs with high single-base discrimination ability. Given that this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, we also demonstrate its application by analyzing miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients.

  13. Simultaneous Characterization of Somatic Events and HPV-18 Integration in a Metastatic Cervical Carcinoma Patient Using DNA and RNA Sequencing

    PubMed Central

    Liang, Winnie S.; Aldrich, Jessica; Nasser, Sara; Kurdoglu, Ahmet; Phillips, Lori; Reiman, Rebecca; McDonald, Jacquelyn; Izatt, Tyler; Christoforides, Alexis; Baker, Angela; Craig, Christine; Egan, Jan B.; Chase, Dana M.; Farley, John H.; Bryce, Alan H.; Stewart, A. Keith; Borad, Mitesh J.; Carpten, John D.; Craig, David W.; Monk, Bradley J.

    2014-01-01

    Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis. PMID:24418928

  14. Relationship between RNA/DNA ratio, growth rate and accumulation of selenium in the cells of wheat leaves under the influence of minerals analcime and trepel.

    PubMed

    Martynenko, O I; Kyrylenko, T K; Zaimenko, N V; Antonyuk, M M; Stepanyugin, A V; Plodnik, D P; Hovorun, D M

    2014-01-01

    We studied specific effects of different doses of natural minerals--analcime (An) and trepel (Tr)--on the growth rate, selenium (Se) content and functional activity of the genome of wheat leaves measured by the RNA/DNA ratio. Our results show that under the influence of An and Tr, especially at low doses (25 mg/100 g sand), there is a significant increase in the content of Se, increased growth rate of leaves of wheat seedlings and decreased RNA/DNA ratio. We have found significant correlations between studied parameters. Our findings suggest that the RNA/DNA ratio can be used as a convenient, reliable indicator of the biological activity of minerals An and Tr, and for quantitative express-estimation of their impact on plant organisms. PMID:25816592

  15. Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway).

    PubMed

    Catarino, Ana I; Cabral, Henrique N; Peeters, Kris; Pernet, Philippe; Punjabi, Usha; Dubois, Philippe

    2008-07-01

    The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the Sørfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The Sørfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions. PMID:18260690

  16. Gene silencing without DNA. rna-mediated cross-protection between viruses

    PubMed Central

    Ratcliff, FG; MacFarlane, SA; Baulcombe, DC

    1999-01-01

    Previously, it was shown that the upper leaves of plants infected with nepoviruses and caulimoviruses are symptom free and contain reduced levels of virus. These leaves are said to be recovered. Recovery is associated with RNA-mediated cross-protection against secondary virus infection. Here, by analyzing plants infected with viruses that are quite distinct from the nepovirus or caulimovirus groups, we demonstrate that this RNA-mediated defense is a general response to virus infection. Upon infection with a tobravirus, plants exhibited RNA-mediated cross-protection and recovery, as occurs in nepovirus-infected plants. However, upon infection with a potexvirus, plants exhibited RNA-mediated cross-protection without recovery. In both instances, a transient gene expression assay showed that RNA-mediated cross-protection was functionally equivalent to post-transcriptional gene silencing. Combined, these data provide direct evidence that post-transcriptional gene silencing of nuclear genes is a manifestation of a natural defense mechanism that is induced by a wide range of viruses. PMID:10402423

  17. Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

    PubMed Central

    Peyrottes, S; Vasseur, J J; Imbach, J L; Rayner, B

    1996-01-01

    Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section. PMID:8657564

  18. Methylated DNA and microRNA in Body Fluids as Biomarkers for Cancer Detection

    PubMed Central

    Ma, Yanning; Wang, Xian; Jin, Hongchuan

    2013-01-01

    Epigenetic alterations including DNA methylation and microRNAs (miRNAs) play important roles in the initiation and progression of human cancers. As the extensively studied epigenetic changes in tumors, DNA methylation and miRNAs are the most potential epigenetic biomarkers for cancer diagnosis. After the identification of circulating cell-free nuclear acids, increasing evidence demonstrated great potential of cell-free epigenetic biomarkers in the blood or other body fluids for cancer detection. PMID:23681012

  19. Structural basis for CRISPR RNA-guided DNA recognition by Cascade

    Microsoft Academic Search

    Matthijs M Jore; Magnus Lundgren; Esther van Duijn; Jelle B Bultema; Edze R Westra; Sakharam P Waghmare; Blake Wiedenheft; Ümit Pul; Reinhild Wurm; Rolf Wagner; Marieke R Beijer; Arjan Barendregt; Kaihong Zhou; Ambrosius P L Snijders; Mark J Dickman; Jennifer A Doudna; Egbert J. Boekema; Albert J. R. Heck; Stan J J Brouns; John van der Oost

    2011-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa

  20. Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

    PubMed Central

    Kapoor, Vikram; Pitkänen, Tarja; Ryu, Hodon; Elk, Michael

    2014-01-01

    The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources. PMID:25326295