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Sample records for macromolecules dna rna

  1. Molecular Biology DNA: The Genetic Macromolecule

    E-print Network

    Molecular Biology Lab 1 DNA: The Genetic Macromolecule (Bacterial Transformation) Although we have begun our Molecular Biology course accepting that DNA is the genetic material of the cell, our first lab of the E.coli genome. The experiment will allow the student to indirectly observe the central dogma

  2. Simultaneous RNA-DNA FISH.

    PubMed

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA. PMID:26721488

  3. Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

    PubMed Central

    2015-01-01

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

  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. Hairpins under tension: RNA versus DNA.

    PubMed

    Bercy, Mathilde; Bockelmann, Ulrich

    2015-11-16

    We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity. PMID:26323319

  7. Cationic Amphiphilic Macromolecule (CAM)-lipid Complexes for Efficient siRNA Gene Silencing

    PubMed Central

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

    2014-01-01

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

  8. RNA-directed DNA methylation

    PubMed Central

    2010-01-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt-long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives. PMID:20421728

  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. Linear and ring DNA macromolecules moderately and strongly confined in nanochannels

    NASA Astrophysics Data System (ADS)

    Cifra, Peter; Benkova, Zuzana; Bleha, Tomas

    2013-03-01

    Understanding the mechanism of DNA extension in nanochannels is necessary for interpretation of experiments in nanofluidic channel devices that are conducted recently not only with linear but also with ring chains. Except reviewing the situation with linear chains we analyze here the experimental results and simulations for the channel-induced extension (linearization) of ring chains. Results of simulations for confined rings indicate that similar transition between moderate and strong confinement as in the case of linear chains exists also for rings. Due to stronger self-avoidance in confined rings the transition and relative chain extension is shifted in comparison to linear DNA. We suggest that similar relation as used in experiments for the extension of linear chains may be used also for circular DNA. For linear DNA in channel relatively stable distinctive events due to chain backfolding, which complicate chain linearization experiments, are analyzed. The abundance of DNA chains folded at the chain ends and in the chain interior was analyzed as a function of the channel width. Z. Benkova, P.Cifra, Macromolecules 45, 2597-2608 (2012) P. Cifra, T.Bleha, Soft Matter 8, 9022-9028 (2012) We acknowledge the support from grant SRDA-0451-11, VEGA grants 2/0093/12 and 2/0079/12 and by the FCT postdoc (Z.B.) co-financed by the Europ. Soc. Found, grant number SFRH/BPD/63568/2009

  11. RNA-Linked DNA Fragments In Vitro*

    PubMed Central

    Sugino, Akio; Okazaki, Reiji

    1973-01-01

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

  12. A DNA enzyme that cleaves RNA

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  13. A lncRNA to repair DNA.

    PubMed

    Lukas, Jiri; Altmeyer, Matthias

    2015-11-01

    Long non-coding RNAs (lncRNAs) have emerged as regulators of various biological processes, but to which extent lncRNAs play a role in genome integrity maintenance is not well understood. In this issue of EMBO Reports, Sharma et al [1] identify the DNA damage-induced lncRNA DDSR1 as an integral player of the DNA damage response (DDR). DDSR1 has both an early role by modulating repair pathway choices, and a later function when it regulates gene expression. Sharma et al [1] thus uncover a dual role for a hitherto uncharacterized lncRNA during the cellular response to DNA damage. PMID:26420434

  14. 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

  15. Image library of biological macromolecules.

    PubMed

    Sühnel, J

    1996-06-01

    An Image Library of Biological Macromolecules is described, which contains image and text files related to structures of biological macromolecules. Currently, the Library has approximately 3000 image files of approximately 300 structures of biological macromolecules whose coordinates are available in the Protein Data Bank and in the Nucleic Acid Database. The entries include all RNA structures, approximately 70 DNA structures, 150 proteins and a few carbohydrates. The Library contains further images of amino acids, of standard and modified nucleotides and of nucleic acid model structures. Each entry consists of an annotation file with bibliographic and sequence information and possibly comments, of a color-coded distance plot and of structure images. Almost all of the images are available both in a mono and in a stereo representation. Standard procedures for generating these images were strictly avoided. Therefore, mixed rendering, coloring and labeling techniques were used extensively. Since May 1995 the Library has a growing division of images in the new Virtual Reality Modeling Language (VRML) format. The Image Library of Biological Macromolecules can be accessed via the World-Wide Web (http://www.imb-jena.de/IMAGE.html). There is a large number of structures determined by experimental and/or modeling techniques which are not intended to be included into the Protein Data Bank or Nucleic Acid Database for some reason. The Image Library could be a repository of these structures and of images of these and other structures of biological macromolecules including structures which are not known at atomic detail. Authors who are willing to make available images or coordinates to the scientific community via the Image Library of Biological Macromolecules are requested to contact the author. PMID:8872391

  16. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  17. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  18. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD) simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted ?-values suggest that C7, G9, dC12, dC15, and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS) P-test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein. PMID:26594643

  19. DNA and RNA Quadruplex-Binding Proteins

    PubMed Central

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

    2014-01-01

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

  20. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

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

    E-print Network

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

    2015-07-21

    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.

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

    SciTech Connect

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

    2014-11-07

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Discrimination of RNA versus DNA by polynucleotide phosphorylase

    PubMed Central

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2013-01-01

    Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also suggested to participate in bacterial DNA transactions. Here we used chimeric polynucleotides, composed of alternating RNA and DNA tracts, to analyze whether and how Mycobacterium smegmatis PNPase discriminates RNA versus DNA during the 3' phosphorolysis reaction. We find that a kinetic block to 3' phosphorolysis of a DNA tract within an RNA polynucleotide is exerted when resection has progressed to the point that a 3' monoribonucleotide flanks the impeding DNA segment. The position of the pause one nucleotide upstream of the first deoxynucleotide encountered is independent of DNA tract length. However, the duration of the pause is affected by DNA tract length, being transient for a single deoxynucleotide and durable when two or more consecutive deoxynucleotides are encountered. Substituting manganese for magnesium as the metal cofactor enables PNPase to “nibble” into the DNA tract. A 3'-phosphate group prevents RNA phosphorolysis when the metal cofactor is magnesium. With manganese, PNPase can resect an RNA 3'-phosphate end, albeit 80-fold slower than a 3'-OH. We discuss the findings in light of the available structures of PNPase and of the archaeal exosome•RNA•phosphate complex and propose a model for catalysis whereby the metal cofactor interacts with the scissile phosphodiester and the penultimate ribose. PMID:23980617

  6. DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA

    E-print Network

    Jacobsen, Steve

    DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS (where H = A, T, or C) sequence contexts and is controlled by at least four DNA methyltransferases

  7. RNA-Linked Nascent DNA Fragments in Escherichia coli*

    PubMed Central

    Sugino, Akio; Hirose, Susumu; Okazaki, Reiji

    1972-01-01

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

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

    E-print Network

    Clote, Peter

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

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

    PubMed Central

    Brabant, F; Acheson, N H

    1995-01-01

    RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion. PMID:7769704

  10. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  11. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  12. Diversity of Endonuclease V: From DNA Repair to RNA Editing

    PubMed Central

    Kuraoka, Isao

    2015-01-01

    Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism. PMID:26404388

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

    E-print Network

    enables RNA molecules to play a central role in many basic biological systems including translation, gene expression, regulation and suppression, viruses, and bio- catalysis.2-4 In addition, the in vitro evolution

  14. RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

    PubMed Central

    Altmann, C R; Solow-Cordero, D E; Chamberlin, M J

    1994-01-01

    In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein. Images PMID:7513426

  15. An Efficient Catalytic DNA that Cleaves L-RNA

    PubMed Central

    Tram, Kha; Xia, Jiaji; Gysbers, Rachel; Li, Yingfu

    2015-01-01

    Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples. PMID:25946137

  16. Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure.

    PubMed

    Takeuchi, Yosuke; Endo, Masayuki; Suzuki, Yuki; Hidaka, Kumi; Durand, Guillaume; Dausse, Eric; Toulmé, Jean-Jacques; Sugiyama, Hiroshi

    2015-12-15

    RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures. PMID:26438892

  17. A brief history of the tree of life Pre DNA the tree of life is made by `organismal biology'

    E-print Network

    Morante, Silvia

    and Transport RNA Translation Proteins #12;Bacterial (prokaryotic) cell Plant (eukaryotic) cell Animal (eukaryotic) cell #12;1. CytoskeletonNucleolus 2. Nucleus 3. Ribosome 4. Vesicle 5. Rough endoplasmic Shigella to Escherichia coli #12;MacromoleculesMacromolecules Cell Membrane DNA RNA Transcription

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

    NASA Astrophysics Data System (ADS)

    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.

  19. 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. PMID:25906017

  20. DNA homologies of ribosomal RNA genes of Neurospora species

    SciTech Connect

    Mukhopadhyay, D.K.; Mimiko, R.; Dutta, S.K.

    1980-01-01

    Ribosomal RNA genes (rDNAs) of Neurospora crassa contain DNA sequences which code for 17S, 5.8S, and 26S rRNAs, in addition to internal and external spacers. As has been reported for many eukaryotes, the DNA sequences which code for 17S, 5.8S, and 26S rRNAs in Neurospora species are probably conserved while the internal and external spacer regions are probably variable sequences. Extensive electron microscopic studies of 45S precursor rRNA of several cold and warm blooded animals confirm that spacer regions vary extensively from species to species. It was desirable to know whether such differences in rDNA sequences exist between Neurospora species. Any such difference should be detectable using standard procedures for DNA homology studies rDNA sequences were isolated from N. crassa mycelial cells using the procedure described previously. The purified rDNA was /sup 3/H-labeled (by nick translation) and reassociated with total DNA isolated from the heterothallic species N. crassa and from three homothalliospecies: N. dodgei, N. lineolata, and N. africana. In addition, /sup 32/P-labeled total DNA of N. crassa was reannealed with unlabeled bulk DNA from N. crassa, N. dodgei, and N. lineolata.

  1. 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.

  2. siRNA-directed DNA Methylation in Plants

    PubMed Central

    Xie, Meng; Yu, Bin

    2015-01-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  3. PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

    E-print Network

    Mosher, Rebecca

    ). Targets of the PolIV-dependent RdDM include 5S rRNA-encoding DNA (rDNA) arrays; regulatory regions with a role of PolIV in the transcription of a DNA template to generate a long RNA precursor of si

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

    ERIC Educational Resources Information Center

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

    2014-01-01

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

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

    PubMed Central

    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

  6. Dicer-independent RNA-directed DNA methylation in Arabidopsis.

    PubMed

    Yang, Dong-Lei; Zhang, Guiping; Tang, Kai; Li, Jingwen; Yang, Lan; Huang, Huan; Zhang, Heng; Zhu, Jian-Kang

    2016-01-01

    RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci. PMID:26642813

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

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

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

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

    PubMed Central

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

    2015-01-01

    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

  9. RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing.

    PubMed Central

    Jones, L; Hamilton, A J; Voinnet, O; Thomas, C L; Maule, A J; Baulcombe, D C

    1999-01-01

    Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X-rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA-DNA interaction that ensures that PTGS is maintained. PMID:10590159

  10. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    PubMed Central

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences. PMID:26500066

  11. 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.

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

    PubMed

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

    2010-01-01

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

  13. 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

  14. Did the Pre-RNA World Rest Upon DNA Molecules?

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  15. The RNA World: Life Before DNA and Protein

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F.

    1993-01-01

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

  16. The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals.

    PubMed

    Zhang, Hao-Ying; Xiong, Jun; Qi, Bao-Ling; Feng, Yu-Qi; Yuan, Bi-Feng

    2015-12-24

    We developed a novel strategy by oxidation-derivatization combined mass spectrometry analysis for the determination of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA. We reported the presence of 5-formylcytosine in RNA of mammals and found that ascorbic acid and hydroquinone can increase the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine in DNA and RNA. PMID:26562407

  17. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

    PubMed Central

    Hamperl, Stephan; Cimprich, Karlene A.

    2014-01-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

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

    PubMed

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

    2014-01-01

    The central dogma of molecular biology, a model that has remained intact for decades, describes the transfer of genetic information from DNA to protein though an RNA intermediate. While recent work has illustrated many exceptions to the central dogma, it is still a common model used to describe and study the relationship between genes and protein products. We investigated understanding of central dogma concepts and found that students are not primed to think about information when presented with the canonical figure of the central dogma. We also uncovered conceptual errors in student interpretation of the meaning of the transcription arrow in the central dogma representation; 36% of students (n = 128; all undergraduate levels) described transcription as a chemical conversion of DNA into RNA or suggested that RNA existed before the process of transcription began. Interviews confirm that students with weak conceptual understanding of information flow find inappropriate meaning in the canonical representation of central dogma. Therefore, we suggest that use of this representation during instruction can be counterproductive unless educators are explicit about the underlying meaning. PMID:26086664

  19. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

    PubMed Central

    Thompson, David B.; Cronican, James J.; Liu, David R.

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

  20. Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells.

    PubMed

    Thompson, David B; Cronican, James J; Liu, David R

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Khodadadi, S.; Roh, J.H.; Kisliuk, A.; Mamontov, E.; Tyagi, M.; Woodson, S.A.; Briber, R.M.; Sokolov, A.P.

    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. PMID:20371332

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

    SciTech Connect

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

    2010-01-01

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

  4. 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.

  5. Role of the CCA Bulge of Prohead RNA of Bacteriophage ø29 in DNA Packaging

    PubMed Central

    Zhao, Wei; Morais, Marc C.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley

    2011-01-01

    Summary The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ø29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. CryoEM-3D reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants. PMID:18778713

  6. A PCR-based approach to assess genomic DNA contamination in RNA: Application to rat RNA samples.

    PubMed

    Padhi, Bhaja K; Singh, Manjeet; Huang, Nicholas; Pelletier, Guillaume

    2016-02-01

    Genomic DNA (gDNA) contamination of RNA samples can lead to inaccurate measurement of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR). We describe an easily adoptable PCR-based method where gDNA contamination in RNA samples is assessed by comparing the amplification of intronic and exonic sequences from a housekeeping gene. Although this alternative assay was developed for rat RNA samples, it could be easily adapted to other species. As a proof of concept, we assessed the effects of detectable gDNA contamination levels on the expression of a few genes that illustrate the importance of RNA quality in acquiring reliable data. PMID:26545322

  7. DNA-, rRNA-and mRNA-based stable isotope probing of aerobic methanotrophs in lake sedimentemi_2415 1..15

    E-print Network

    Saleska, Scott

    DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sedimentemi_2415 oxic sedi- ment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane-labelled DNA and RNA from unlabelled nucleic acids. The incubation with 13 CH4 was performed over a 4-day time

  8. Cytoplasmic RNA sequences complementary to cloned chick delta-crystallin cDNA show size heterogeneity.

    PubMed Central

    Bower, D J; Errington, L H; Wainwright, N R; Sime, C; Morris, S; Clayton, R M

    1982-01-01

    Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes. Images Fig. 1. Fig. 2. Fig. 4. PMID:6282266

  9. Programmed self-assembly of DNA/RNA for biomedical applications

    NASA Astrophysics Data System (ADS)

    Wang, Pengfei

    Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

  10. The Roads to and from the RNA World

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  11. 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.

  12. 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

  13. Symmetry and structure of RNA and DNA triple helices.

    PubMed

    Raghunathan, G; Miles, H T; Sasisekharan, V

    1995-09-01

    Despite wide interest in nucleic acid triple helices, there has been no stereochemically satisfactory structure of an RNA triple helix in atomic detail. AN RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244, pp. 99-101; S. Arnott, P. J. Bond, E. Selsing, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3, pp. 2459-2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new model for an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3-11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from the introduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455-462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu, H. T. Miles, K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1, pp. 11-12]. The above triple helices have Watson-Crick-Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16, pp. 907-916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson-Crick-reverse Hoogsteen, Donohue [J. Donohue (1953) Proceedings of the National Academy of Science USA, Vol. 39, pp. 470-475] (reverse Watson-Crick)-Hoogsteen, and Donohue-reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain. PMID:7545446

  14. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity

    PubMed Central

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M.

    2015-01-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA–RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA–RNA and RNA–RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA–RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  15. 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.

  16. 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

  17. Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes

    PubMed Central

    Schmitz, Kerstin-Maike; Mayer, Christine; Postepska, Anna; Grummt, Ingrid

    2010-01-01

    Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoter-associated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation. PMID:20952535

  18. 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

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

    PubMed Central

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

    2008-01-01

    RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5?-PO4 single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65°C, AppRNA was predominantly ligated to a circular product. In contrast, at 35°C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases. PMID:18829718

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

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

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

    SciTech Connect

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

    2011-07-25

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

  2. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  4. A rapid procedure to detect in situ DNA/RNA hybrids

    SciTech Connect

    Reddy, A.R.; Sofer, W.

    1981-12-15

    A new method was developed for detecting DNA/RNA hydrids formed in situ using anti-DNA/RNA antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesized in vitro from cloned Drosophila histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.

  5. A comparison of RNA with DNA in template-directed synthesis

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    E-print Network

    Pang, Bo

    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. ...

  7. Comparison of the Prognostic Utility of the Diverse Molecular Data among lncRNA, DNA Methylation, microRNA, and mRNA across Five Human Cancers

    PubMed Central

    Xu, Li; Fengji, Liang; Changning, Liu; Liangcai, Zhang; Yinghui, Li; Yu, Li; Shanguang, Chen; Jianghui, Xiong

    2015-01-01

    Introduction Advances in high-throughput technologies have generated diverse informative molecular markers for cancer outcome prediction. Long non-coding RNA (lncRNA) and DNA methylation as new classes of promising markers are emerging as key molecules in human cancers; however, the prognostic utility of such diverse molecular data remains to be explored. Materials and Methods We proposed a computational pipeline (IDFO) to predict patient survival by identifying prognosis-related biomarkers using multi-type molecular data (mRNA, microRNA, DNA methylation, and lncRNA) from 3198 samples of five cancer types. We assessed the predictive performance of both single molecular data and integrated multi-type molecular data in patient survival stratification, and compared their relative importance in each type of cancer, respectively. Survival analysis using multivariate Cox regression was performed to investigate the impact of the IDFO-identified markers and traditional variables on clinical outcome. Results Using the IDFO approach, we obtained good predictive performance of the molecular datasets (bootstrap accuracy: 0.71–0.97) in five cancer types. Impressively, lncRNA was identified as the best prognostic predictor in the validated cohorts of four cancer types, followed by DNA methylation, mRNA, and then microRNA. We found the incorporating of multi-type molecular data showed similar predictive power to single-type molecular data, but with the exception of the lncRNA + DNA methylation combinations in two cancers. Survival analysis of proportional hazard models confirmed a high robustness for lncRNA and DNA methylation as prognosis factors independent of traditional clinical variables. Conclusion Our study provides insight into systematically understanding the prognostic performance of diverse molecular data in both single and aggregate patterns, which may have specific reference to subsequent related studies. PMID:26606135

  8. Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection.

    PubMed

    Shi, Chao; Shen, Xiaotong; Niu, Shuyan; Ma, Cuiping

    2015-11-01

    RNA detection has become one of the most robust parts in molecular biology, medical diagnostics and drug discovery. Conventional RNA detection methods involve an extra reverse transcription step, which limits their further application for RNA rapid detection. We herein report a novel finding that Bst and Klenow DNA polymerases possess innate reverse transcriptase activities, so that the reverse transcription step and next amplification reaction can be combined to one step in isothermal RNA detection. We have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 125-nt length by real time RT-PCR and polyacrylamide gel electrophoresis (PAGE). Our findings will spur the development of a myriad of simple and easy to use RNA detection technologies for isothermal RNA direct detection. This will just meet the future needs of bioanalysis and clinical diagnosis to RNA rapid detection in POC settings and inspection and quarantine. PMID:26474356

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

    E-print Network

    Dekker, Nynke

    random- sequence dsDNA in bending, stretching, and torsional behaviors; Z-DNA to be at least three. These calculations recovered the experimental bending persistence length of dsRNA within the error of the simulations modulus relative to dsDNA. Further blind predictions of helix torsional properties, however, exposed

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

    E-print Network

    Tripathi, Tripti

    2007-01-01

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

  11. Nuclear (DNA, RNA, histone and non-histone protein) and nucleolar changes during growth and senescence of may apple leaves.

    PubMed

    Bhattacharya, P K; Pappelis, A J; Lee, S C; BeMiller, J N; Karagiannis, C S

    1996-12-20

    Quantitative interference microscopy was used to determine changes in nuclear and nucleolar indices (dry mass and cross-sectional area) in upper and lower epidermal cells and adjacent leaf-margin hair cells of the May apple (Podophyllum peltatum L.) leaves over a 42-day period (after leaves emerged above the ground litter). These indices decreased in a highly correlated manner. A ploidy variation may exist between epidermal cells and leaf-margin hair cells. Using the leaf-margin hair cells model, six nuclear macromolecule indices (total nucleic acid, DNA, RNA, total nuclear protein, histone and non-histone protein), nuclear volume, nucleolar volume and perinucleolar volume (measured using quantitative epifluorescence-phase contrast microscopy) all declined with age (42-day study) in a highly correlated manner. The degeneration of the nucleus and nucleolus in the three leaf locations studied followed the patterns observed for programmed cellular senescence and death (necrosis) in epidermal cells of onion leaf bases (stored tissue; leaf bases did not contain chlorophyll) and human epithelial cells (buccal; cervical). We conclude that the epidermal cells and leaf-margin hair cells from green leaves of the May Apple are ideal for the study of programmed cell senescence and death in plants, especially for the partitioning of this process into the study of: the point-of-no-return (solubilization of the karyoskeleton and loss of non-histone proteins and RNA associated with the karyoskeleton from the nucleus); nuclear pycnosis (loss of nuclear dry mass and volume and loss of nuclear internal support structure); chromatin condensation, margination along the inner nuclear envelope; and DNA-histone degeneration; degeneration of the nucleolus and loss of the perinucleolar zone of exclusion. The characterization of chlorenchyma cells during the 42-day period should now be undertaken (leaf senescence as indicated by the beginning of yellowing about 35 days after emergence) to determine whether these cells with functional chloroplasts undergo nuclear changes like those lacking functional chloroplasts. PMID:9080390

  12. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    PubMed Central

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-01-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation. PMID:26461456

  13. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin.

    PubMed

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-01-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation. PMID:26461456

  14. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    NASA Astrophysics Data System (ADS)

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-10-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

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

    PubMed

    Kim, Sun-Hyung; Hamada, Tatsuro

    2005-12-01

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

  16. Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

    PubMed Central

    Szabat, Marta; Pedzinski, Tomasz; Czapik, Tomasz; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes. PMID:26579720

  17. Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei 1

    PubMed Central

    Guilfoyle, Tom J.

    1976-01-01

    DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to ?-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 ?g/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of ?-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates. Images PMID:16659696

  18. Transcription of DNA from the 70S RNA of Rous Sarcoma Virus II. Structure of a 4S RNA Primer

    PubMed Central

    Faras, A. J.; Dahlberg, J. E.; Sawyer, R. C.; Harada, F.; Taylor, J. M.; Levinson, W. E.; Bishop, J. M.; Goodman, H. M.

    1974-01-01

    The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways—by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5? terminus and CpCpAOH at the 3? terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (?p, ?p, Cp) Gp (possibly in place of the characteristic sequence GprTp?pCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro. Images PMID:4132920

  19. Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana

    PubMed Central

    Woo, Hye Ryun; Richards, Eric J

    2008-01-01

    Background DNA methylation is an important biochemical mark that silences repetitive sequences, such as transposons, and reinforces epigenetic gene expression states. An important class of repetitive genes under epigenetic control in eukaryotic genomes encodes ribosomal RNA (rRNA) transcripts. The ribosomal genes coding for the 45S rRNA precursor of the three largest eukaryotic ribosomal RNAs (18S, 5.8S, and 25–28S) are found in nucleolus organizer regions (NORs), comprised of hundreds to thousands of repeats, only some of which are expressed in any given cell. An epigenetic switch, mediated by DNA methylation and histone modification, turns rRNA genes on and off. However, little is known about the mechanisms that specify and maintain the patterns of NOR DNA methylation. Results Here, we explored the extent of naturally-occurring variation in NOR DNA methylation among accessions of the flowering plant Arabidopsis thaliana. DNA methylation in coding regions of rRNA genes was positively correlated with copy number of 45S rRNA gene and DNA methylation in the intergenic spacer regions. We investigated the inheritance of NOR DNA methylation patterns in natural accessions with hypomethylated NORs in inter-strain crosses and defined three different categories of inheritance in F1 hybrids. In addition, subsequent analysis of F2 segregation for NOR DNA methylation patterns uncovered different patterns of inheritance. We also revealed that NOR DNA methylation in the Arabidopsis accession Bor-4 is influenced by the vim1-1 (variant in methylation 1-1) mutation, but the primary effect is specified by the NORs themselves. Conclusion Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation. However, the inheritance of NOR DNA methylation suggests the operation of a diverse set of mechanisms, including inheritance of parental methylation patterns, reconfiguration of parental NOR DNA methylation, and the involvement of trans-acting modifiers. PMID:18783613

  20. Mimicking the First Step of RNA Splicing: An Artificial DNA Enzyme Can Synthesize Branched RNA Using an Oligonucleotide Leaving Group as a

    E-print Network

    Silverman, Scott K.

    Mimicking the First Step of RNA Splicing: An Artificial DNA Enzyme Can Synthesize Branched RNA mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide

  1. MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response

    PubMed Central

    Pothof, Joris; Verkaik, Nicole S; van IJcken, Wilfred; Wiemer, Erik A C; Ta, Van T B; van der Horst, Gijsbertus T J; Jaspers, Nicolaas G J; van Gent, Dik C; Hoeijmakers, Jan H J; Persengiev, Stephan P

    2009-01-01

    DNA damage provokes DNA repair, cell-cycle regulation and apoptosis. This DNA-damage response encompasses gene-expression regulation at the transcriptional and post-translational levels. We show that cellular responses to UV-induced DNA damage are also regulated at the post-transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA-mediated gene-silencing inhibition by knocking down essential components of the microRNA-processing pathway (Dicer and Ago2). UV damage triggered a cell-cycle-dependent relocalization of Ago2 into stress granules and various microRNA-expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV-responsive microRNA expression was only partially ATM/ATR independent. Both microRNA-expression changes and stress-granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA-mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV-inducible miR-16 that downregulates checkpoint-gene CDC25a and regulates cell proliferation. We conclude that microRNA-mediated gene regulation adds a new dimension to the DNA-damage response. PMID:19536137

  2. 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

  3. Factor-independent transcription pausing caused by recognition of the RNA–DNA hybrid sequence

    PubMed Central

    Bochkareva, Aleksandra; Yuzenkova, Yulia; Tadigotla, Vasisht R; Zenkin, Nikolay

    2012-01-01

    Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined by the ability of the elongating RNA polymerase to recognize the sequence of the RNA–DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly ‘senses' the shape and/or identity of base pairs of the RNA–DNA hybrid. Recognition of the RNA–DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through ‘in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA–DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation. PMID:22124324

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    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.

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

    PubMed

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

    2008-01-01

    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. PMID:18834537

  6. Evolution from DNA to RNA Recognition by the bI3 LAGLIDADG Maturase

    SciTech Connect

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

    2005-01-01

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

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

    E-print Network

    Bong, Dennis

    Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery- 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

  8. On the path to genetic novelties: insights from programmed DNA elimination and RNA splicing.

    PubMed

    Catania, Francesco; Schmitz, Jürgen

    2015-01-01

    Understanding how genetic novelties arise is a central goal of evolutionary biology. To this end, programmed DNA elimination and RNA splicing deserve special consideration. While programmed DNA elimination reshapes genomes by eliminating chromatin during organismal development, RNA splicing rearranges genetic messages by removing intronic regions during transcription. Small RNAs help to mediate this class of sequence reorganization, which is not error-free. It is this imperfection that makes programmed DNA elimination and RNA splicing excellent candidates for generating evolutionary novelties. Leveraging a number of these two processes' mechanistic and evolutionary properties, which have been uncovered over the past years, we present recently proposed models and empirical evidence for how splicing can shape the structure of protein-coding genes in eukaryotes. We also chronicle a number of intriguing similarities between the processes of programmed DNA elimination and RNA splicing, and highlight the role that the variation in the population-genetic environment may play in shaping their target sequences. PMID:26140477

  9. 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...

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

    PubMed Central

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

    2009-01-01

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

  11. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

    SciTech Connect

    Yang, Cai-Guang; Yi, Chengqi; Duguid, Erica M.; Sullivan, Christopher T.; Jian, Xing; Rice, Phoebe A.; He, Chuan

    2008-09-26

    Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.

  12. 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

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

    DOEpatents

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

    1995-04-11

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

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

    DOEpatents

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

    1995-01-01

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

  15. Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system.

    PubMed

    Stuckey, Ruth; García-Rodríguez, Néstor; Aguilera, Andrés; Wellinger, Ralf Erik

    2015-05-01

    DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-? and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis. PMID:25902524

  16. Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

    PubMed Central

    Stuckey, Ruth; García-Rodríguez, Néstor; Aguilera, Andrés; Wellinger, Ralf Erik

    2015-01-01

    DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-? and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop–mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis. PMID:25902524

  17. 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

  18. Direct Detection of Genomic DNA by Enzymatically Amplified SPR Imaging Measurements of RNA Microarrays

    E-print Network

    in Scheme 1. A single-stranded RNA microarray is exposed to a solution that contains a complementary ssDNADirect Detection of Genomic DNA by Enzymatically Amplified SPR Imaging Measurements of RNA Microarrays Terry T. Goodrich, Hye Jin Lee, and Robert M. Corn* Department of Chemistry, Uni

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi.

    1989-12-26

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

  1. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus.

    PubMed

    Miller, Eric

    2016-01-01

    Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5' and 3' untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts. PMID:26187203

  2. 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

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

    PubMed Central

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

    2011-01-01

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

  4. Phage T4-induced DNA breaks activate a tRNA repair-defying anticodon nuclease.

    PubMed

    Bitton, Lital; Klaiman, Daniel; Kaufmann, Gabriel

    2015-09-01

    The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi?RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction. PMID:26031711

  5. 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

  6. 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

  7. Molecular basis of human telomere DNA/RNA structure and its potential application.

    PubMed

    Xu, Yan; Suzuki, Yuta; Kaminaga, Kuniyuki; Komiyama, Makoto

    2009-01-01

    Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. However, structure and function of the telomeric RNA in chromosome ends have not yet been elucidated. Using a combination of NMR, CD and MALDI-TOFMS experiments, we have demonstrated that human telomere RNA can form a parallel G-quadruplex structure. Furthermore, we successfully found for the first time that human telomere DNA and RNA sequence can form a DNA-RNA hybrid type G-quadruplex structure based on click chemistry approach. Telomerase or its telomere DNA substrate is also known to present a specific target in discovering anticancer agents. Recently, we developed a structure-based approach to sequence-specific cleaving of human telomeric DNA by G-quadruplex formation. These results not only provide valuable information to allow understanding of the roles of human telomeric RNA in telomere biology, but also serve as a start step for developing new anti-cancer reagent. PMID:19749261

  8. Viral nanomotors for packaging of dsDNA and dsRNA

    PubMed Central

    Guo, Peixuan; Lee, Tae Jin

    2007-01-01

    While capsid proteins are assembled around single-stranded genomic DNA or RNA in rod-shaped viruses, the lengthy double-stranded genome of other viruses is packaged forcefully within a preformed protein shell. This entropically unfavourable DNA or RNA packaging is accomplished by an ATP-driven viral nanomotor, which is mainly composed of two components, the oligomerized channel and the packaging enzymes. This intriguing DNA or RNA packaging process has provoked interest among virologists, bacteriologists, biochemists, biophysicists, chemists, structural biologists and computational scientists alike, especially those interested in nanotechnology, nanomedicine, AAA+ family proteins, energy conversion, cell membrane transport, DNA or RNA replication and antiviral therapy. This review mainly focuses on the motors of double-stranded DNA viruses, but double-stranded RNA viral motors are also discussed due to interesting similarities. The novel and ingenious configuration of these nanomotors has inspired the development of biomimetics for nanodevices. Advances in structural and functional studies have increased our understanding of the molecular basis of biological movement to the point where we can begin thinking about possible applications of the viral DNA packaging motor in nanotechnology and medical applications. PMID:17501915

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-07-01

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

  11. MEG3 long noncoding RNA regulates the TGF-? pathway genes through formation of RNA–DNA triplex structures

    PubMed Central

    Mondal, Tanmoy; Subhash, Santhilal; Vaid, Roshan; Enroth, Stefan; Uday, Sireesha; Reinius, Björn; Mitra, Sanhita; Mohammed, Arif; James, Alva Rani; Hoberg, Emily; Moustakas, Aristidis; Gyllensten, Ulf; Jones, Steven J.M.; Gustafsson, Claes M; Sims, Andrew H; Westerlund, Fredrik; Gorab, Eduardo; Kanduri, Chandrasekhar

    2015-01-01

    Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-? pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-? genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA–DNA triplex formation. We have found that RNA–DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-? pathway genes. Our findings suggest that RNA–DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs. PMID:26205790

  12. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

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

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

    NASA Astrophysics Data System (ADS)

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

    2002-05-01

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

  14. 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

  15. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    SciTech Connect

    Pallan, Pradeep S.; Egli, Martin

    2009-06-17

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

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

    NASA Astrophysics Data System (ADS)

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

    2001-12-01

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

  19. Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    E-print Network

    Pikaard, Craig

    Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants Investigacion Cienti´ficas, Madrid, Spain Abstract In eukaryotes, 45S rRNA genes are arranged in tandem arrays the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants

  20. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 communitie...

  2. Crystallization of Macromolecules

    PubMed Central

    Friedmann, David; Messick, Troy; Marmorstein, Ronen

    2014-01-01

    X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

  3. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    DOEpatents

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

    2008-12-23

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

  4. 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

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

    PubMed

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

    2014-11-10

    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

  6. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

    PubMed

    Karmakar, Saswata; Harcourt, Emily M; Hewings, David S; Scherer, Florian; Lovejoy, Alexander F; Kurtz, David M; Ehrenschwender, Thomas; Barandun, Luzi J; Roost, Caroline; Alizadeh, Ash A; Kool, Eric T

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37?°C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

  7. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37?°C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  8. Abnormal rapid non-linear RNA production induced by T7 RNA polymerase in the absence of an exogenous DNA template

    NASA Astrophysics Data System (ADS)

    Kakimoto, Y.; Fujinuma, A.; Fujita, S.; Kikuchi, Y.; Umekage, S.

    2015-02-01

    Although recombinant T7 RNA polymerase is commonly used for in vitro RNA synthesis, several reports have pointed out that T7 RNA polymerase can also induce RNA-directed RNA polymerization or replication. In addition, here we show a new aberrant transcription when using T7 RNA polymerase. This polymerization was observed in the presence of both ribonucleotides and a purchasable T7 RNA polymerase, Thermo T7 RNA polymerase, as well as in the absence of an exogenous DNA template. This cryptic RNA production was detectable after several hours of incubation and was inhibited by adding DNase I. These findings suggested that some contaminated DNA along with the Thermo stable T7 RNA polymerase could be used as template DNA. However, to our surprise, RNA production showed a rapid non-linear increase. This finding strongly indicated that a self-replication cycle emerged from the RNA-directed polymerization or replication by T7 RNA polymerase, triggering the abnormal explosive increase.

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

    PubMed

    Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

    2014-06-01

    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

  10. 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

  11. Efficient Automated Solid-Phase Synthesis of DNA and RNA 5'-Triphosphates.

    PubMed

    Sarac, Ivo; Meier, Chris

    2015-11-01

    A fast, high-yielding and reliable method for the synthesis of DNA- and RNA 5'-triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite was coupled to the 5'-end. Oxidation of the formed 5'-phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5'-cycloSal-oligonucleotides. Reaction of the support-bonded 5'-cycloSal-oligonucleotide with pyrophosphate yielded the corresponding 5'-triphosphates. The 5'-triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer. PMID:26517040

  12. 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

  13. 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)

  14. 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

  15. Long-term storage of DNA-free RNA for use in vaccine studies.

    PubMed

    Jones, Kathryn L; Drane, Debbie; Gowans, Eric J

    2007-11-01

    RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved. PMID:18072597

  16. 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

  17. The distinctive cellular responses to DNA strand breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA transcription.

    PubMed

    Sakasai, Ryo; Iwabuchi, Kuniyoshi

    2016-01-01

    Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step, resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to secondary strand breaks when they collide with the DNA replication and RNA transcription machinery. DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents. In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to, transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA damage mediated by DNA replication and RNA transcription. PMID:26616758

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

    PubMed Central

    Lafrance-Vanasse, Julien

    2014-01-01

    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 Watson-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

  19. 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

  20. Rapid determination of enantiomeric ratio using fluorescent DNA or RNA aptamers.

    PubMed

    Null, Eric L; Lu, Yi

    2010-02-01

    The natural chirality of DNA and RNA aptamers has been used to develop fluorescent agents to determine the enantiomeric ratio of adenosine and arginine, respectively. The quantification is based on structure-switching DNA or RNA aptamers labeled with fluorophore and quencher, allowing chiral detection down to 0.1 : 99.9 (L : D) for arginine after calibration. Such a method provides a general platform for simple, low-cost and high throughput detection and quantification of chirality of a broad range of molecules. PMID:20098779

  1. Not just "a clever way to detect whether DNA really made RNA": The invention of DNA-RNA hybridization and its outcome.

    PubMed

    Fisher, Susie

    2015-10-01

    The invention of DNA-RNA hybridization in 1960 by Ben Hall and Sol Spiegelman had a powerful impact on the theory and discourse of molecular biology. Yet, despite its importance, the story of this invention has barely been told. Hybridization allowed biologists to bridge the theoretical realm and the material world of organisms, to correlate a hypothetical concept of biological information transfer with a mechanism capable of making an RNA copy of DNA. During the early 1960s, Spiegelman and coworkers employed hybridization to investigate the origin of RNAs found in cells. They operationally defined messenger RNA and elucidated several aspects of genome organization. For Spiegelman, this was the culmination of his longstanding interest in the mechanism of enzyme/protein synthesis; for Hall, it was the beginning of a successful career in genetics. Other scientists immediately recognized the power of the technique and introduced improvements. In 1965, Gillespie and Spiegelman combined several modifications and described a procedure for hybridization that became standard. Since the 1970s, it has become an essential tool in biology and in biotechnology, and a core component in molecular techniques such as DNA microarrays. Notwithstanding its current success, the inventors' names have disappeared from the literature. This curiosity is discussed. PMID:26209888

  2. Profiles of piRNA abundances at emerging or established piRNA loci are determined by local DNA sequences.

    PubMed

    de Vanssay, Augustin; Bougé, Anne-Laure; Boivin, Antoine; Hermant, Catherine; Teysset, Laure; Delmarre, Valérie; Ronsseray, Stéphane; Antoniewski, Christophe

    2013-08-01

    Piwi-interacting RNAs (piRNAs) ensure transposable element silencing in Drosophila, thereby preserving genome integrity across generations. Primary piRNAs arise from the processing of long RNA transcripts produced in the germ line by a limited number of telomeric and pericentromeric loci. Primary piRNAs bound to the Argonaute protein Aubergine then drive the production of secondary piRNAs through the "ping-pong" amplification mechanism that involves an interplay with piRNAs bound to the Argonaute protein Argonaute-3. We recently discovered that clusters of P-element-derived transgenes produce piRNAs and mediate silencing of homologous target transgenes in the female germ line. We also demonstrated that some clusters are able to convert other homologous inactive transgene clusters into piRNA-producing loci, which then transmit their acquired silencing capacity over generations. This paramutation phenomenon is mediated by maternal inheritance of piRNAs homologous to the transgenes. Here we further mined our piRNA sequencing data sets generated from various strains carrying transgenes with partial sequence homology at distinct genomic sites. This analysis revealed that same sequences in different genomic contexts generate highly similar profiles of piRNA abundances. The strong tendency of piRNAs for bearing a U at their 5' end has long been recognized. Our observations support the notion that, in addition, the relative frequencies of Drosophila piRNAs are locally determined by the DNA sequence of piRNA loci. PMID:23880829

  3. Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences.

    PubMed

    Sanchez-Sandoval, Eugenia; Diaz-Quezada, Corina; Velazquez, Gilberto; Arroyo-Navarro, Luis F; Almanza-Martinez, Norineli; Trasviña-Arenas, Carlos H; Brieba, Luis G

    2015-09-01

    Three proteins phylogenetically grouped with proteins from the T7 replisome localize to yeast mitochondria: DNA polymerase ? (Mip1), mitochondrial RNA polymerase (Rpo41), and a single-stranded binding protein (Rim1). Human and T7 bacteriophage RNA polymerases synthesize primers for their corresponding DNA polymerases. In contrast, DNA replication in yeast mitochondria is explained by two models: a transcription-dependent model in which Rpo41 primes Mip1 and a model in which double stranded breaks create free 3' OHs that are extended by Mip1. Herein we found that Rpo41 transcribes RNAs that can be extended by Mip1 on single and double-stranded DNA. In contrast to human mitochondrial RNA polymerase, which primes DNA polymerase ? using transcripts from the light-strand and heavy-strand origins of replication, Rpo41 primes Mip1 at replication origins and promoter sequences in vitro. Our results suggest that in ori1, short transcripts serve as primers, whereas in ori5 an RNA transcript longer than 29 nucleotides is used as primer. PMID:26184436

  4. do_x3dna: a tool to analyze structural fluctuations of dsDNA or dsRNA from molecular dynamics simulations.

    PubMed

    Kumar, Rajendra; Grubmüller, Helmut

    2015-08-01

    The do_x3dna package has been developed to analyze the structural fluctuations of DNA or RNA during molecular dynamics simulations. It extends the capability of the 3DNA package to GROMACS MD trajectories and includes new methods to calculate the global-helical axis of DNA and bending fluctuations during simulations. The package also includes a Python module dnaMD to perform and visualize statistical analyses of complex data obtained from the trajectories. PMID:25838463

  5. Travel depth, a new shape descriptor for macromolecules: application to ligand binding.

    PubMed

    Coleman, Ryan G; Sharp, Kim A

    2006-09-22

    Depth is a term frequently applied to the shape and surface of macromolecules, describing for example the grooves in DNA, the shape of an enzyme active site, or the binding site for a small molecule in a protein. Yet depth is a difficult property to define rigorously in a macromolecule, and few computational tools exist to quantify this notion, to visualize it, or analyze the results. We present our notion of travel depth, simply put the physical distance a solvent molecule would have to travel from a surface point to a suitably defined reference surface. To define the reference surface, we use the limiting form of the molecular surface with increasing probe size: the convex hull. We then present a fast, robust approximation algorithm to compute travel depth to every surface point. The travel depth is useful because it works for pockets of any size and complexity. It also works for two interesting special cases. First, it works on the grooves in DNA, which are unbounded in one direction. Second, it works on the case of tunnels, that is pockets that have no "bottom", but go through the entire macromolecule. Our algorithm makes it straightforward to quantify discussions of depth when analyzing structures. High-throughput analysis of macromolecule depth is also enabled by our algorithm. This is demonstrated by analyzing a database of protein-small molecule binding pockets, and the distribution of bound magnesium ions in RNA structures. These analyses show significant, but subtle effects of depth on ligand binding localization and strength. PMID:16934837

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

    SciTech Connect

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

    2012-10-23

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

  7. Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

    PubMed Central

    Bardeesy, N; Pelletier, J

    1998-01-01

    The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein. PMID:9512553

  8. mRNA and DNA selection via protein multimerization: YB-1 as a case study

    PubMed Central

    Kretov, Dmitry A.; Curmi, Patrick A.; Hamon, Loic; Abrakhi, Sanae; Desforges, Bénédicte; Ovchinnikov, Lev P.; Pastré, David

    2015-01-01

    Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance. PMID:26271991

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

    PubMed Central

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

    2006-01-01

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

  10. RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

    NASA Technical Reports Server (NTRS)

    Ordoukhanian, Phillip; Joyce, Gerald F.

    2002-01-01

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

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

    PubMed

    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. PMID:25497230

  12. Rolling circle amplification detection of RNA and DNA

    DOEpatents

    Christian, Allen T.; Pattee, Melissa S.; Attix, Cristina M.; Tucker, James D.

    2004-08-31

    Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

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

    E-print Network

    Lohman, Timothy M.

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

  14. High-throughput prediction of RNA, DNA and protein binding regions mediated by intrinsic disorder.

    PubMed

    Peng, Zhenling; Kurgan, Lukasz

    2015-10-15

    Intrinsically disordered proteins and regions (IDPs and IDRs) lack stable 3D structure under physiological conditions in-vitro, are common in eukaryotes, and facilitate interactions with RNA, DNA and proteins. Current methods for prediction of IDPs and IDRs do not provide insights into their functions, except for a handful of methods that address predictions of protein-binding regions. We report first-of-its-kind computational method DisoRDPbind for high-throughput prediction of RNA, DNA and protein binding residues located in IDRs from protein sequences. DisoRDPbind is implemented using a runtime-efficient multi-layered design that utilizes information extracted from physiochemical properties of amino acids, sequence complexity, putative secondary structure and disorder and sequence alignment. Empirical tests demonstrate that it provides accurate predictions that are competitive with other predictors of disorder-mediated protein binding regions and complementary to the methods that predict RNA- and DNA-binding residues annotated based on crystal structures. Application in Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila melanogaster proteomes reveals that RNA- and DNA-binding proteins predicted by DisoRDPbind complement and overlap with the corresponding known binding proteins collected from several sources. Also, the number of the putative protein-binding regions predicted with DisoRDPbind correlates with the promiscuity of proteins in the corresponding protein-protein interaction networks. Webserver: http://biomine.ece.ualberta.ca/DisoRDPbind/. PMID:26109352

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

    E-print Network

    Walter, Nils G.

    to advance our understanding of the central dogma of molecular biology that DNA makes RNA makes protein of nucleic acids in the biology of life has sky- rocketed. For 50 years, Biopolymers as a journal has aimed. CASE Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, NJ 08854 NILS G

  16. Water and Ion Binding Around RNA and DNA (C,G) Oligomers

    E-print Network

    Westhof, Eric

    Water and Ion Binding Around RNA and DNA (C,G) Oligomers Pascal Auffinger and Eric Westhof times of water molecules in the ®rst hydration shell of the helices range from 0.5 to 1 ns, in good agreement with available experimental data. Such water molecules are essentially found in the vicinity

  17. Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer.

    PubMed

    Bersani, Francesca; Lee, Eunjung; Kharchenko, Peter V; Xu, Andrew W; Liu, Mingzhu; Xega, Kristina; MacKenzie, Olivia C; Brannigan, Brian W; Wittner, Ben S; Jung, Hyunchul; Ramaswamy, Sridhar; Park, Peter J; Maheswaran, Shyamala; Ting, David T; Haber, Daniel A

    2015-12-01

    Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture. PMID:26575630

  18. High-throughput prediction of RNA, DNA and protein binding regions mediated by intrinsic disorder

    PubMed Central

    Peng, Zhenling; Kurgan, Lukasz

    2015-01-01

    Intrinsically disordered proteins and regions (IDPs and IDRs) lack stable 3D structure under physiological conditions in-vitro, are common in eukaryotes, and facilitate interactions with RNA, DNA and proteins. Current methods for prediction of IDPs and IDRs do not provide insights into their functions, except for a handful of methods that address predictions of protein-binding regions. We report first-of-its-kind computational method DisoRDPbind for high-throughput prediction of RNA, DNA and protein binding residues located in IDRs from protein sequences. DisoRDPbind is implemented using a runtime-efficient multi-layered design that utilizes information extracted from physiochemical properties of amino acids, sequence complexity, putative secondary structure and disorder and sequence alignment. Empirical tests demonstrate that it provides accurate predictions that are competitive with other predictors of disorder-mediated protein binding regions and complementary to the methods that predict RNA- and DNA-binding residues annotated based on crystal structures. Application in Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila melanogaster proteomes reveals that RNA- and DNA-binding proteins predicted by DisoRDPbind complement and overlap with the corresponding known binding proteins collected from several sources. Also, the number of the putative protein-binding regions predicted with DisoRDPbind correlates with the promiscuity of proteins in the corresponding protein–protein interaction networks. Webserver: http://biomine.ece.ualberta.ca/DisoRDPbind/ PMID:26109352

  19. Endogenous Small RNA Mediates Meiotic Silencing of a Novel DNA Transposon

    PubMed Central

    Wang, Yizhou; Smith, Kristina M.; Taylor, John W.; Freitag, Michael; Stajich, Jason E.

    2015-01-01

    Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory “wild-type” strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference?silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element. PMID:26109355

  20. Non-Coding RNA: Sequence-Specific Guide for Chromatin Modification and DNA Damage Signaling

    PubMed Central

    Francia, Sofia

    2015-01-01

    Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair. PMID:26617633

  1. Downstream DNA tension regulates the stability of the T7 RNA polymerase initiation complex.

    PubMed

    Skinner, Gary M; Kalafut, Bennett S; Visscher, Koen

    2011-02-16

    Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the "scrunching" model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

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

    SciTech Connect

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    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.

  3. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    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. DNA and RNA sequencing by nanoscale reading through programmable electrophoresis and nanoelectrode-gated tunneling and dielectric detection

    DOEpatents

    Lee, James W.; Thundat, Thomas G.

    2005-06-14

    An apparatus and method for performing nucleic acid (DNA and/or RNA) sequencing on a single molecule. The genetic sequence information is obtained by probing through a DNA or RNA molecule base by base at nanometer scale as though looking through a strip of movie film. This DNA sequencing nanotechnology has the theoretical capability of performing DNA sequencing at a maximal rate of about 1,000,000 bases per second. This enhanced performance is made possible by a series of innovations including: novel applications of a fine-tuned nanometer gap for passage of a single DNA or RNA molecule; thin layer microfluidics for sample loading and delivery; and programmable electric fields for precise control of DNA or RNA movement. Detection methods include nanoelectrode-gated tunneling current measurements, dielectric molecular characterization, and atomic force microscopy/electrostatic force microscopy (AFM/EFM) probing for nanoscale reading of the nucleic acid sequences.

  5. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    PubMed Central

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  6. 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

  7. Inheritance of silent rDNA chromatin is mediated by PARP1 via noncoding RNA.

    PubMed

    Guetg, Claudio; Scheifele, Fabian; Rosenthal, Florian; Hottiger, Michael O; Santoro, Raffaella

    2012-03-30

    Faithful propagation of specific chromatin states requires re-establishment of epigenetic marks after every cell division. How the original epigenetic signature is inherited after disruption during DNA replication is still poorly understood. Here, we show that the poly(ADP-ribose)-polymerase-1 (PARP1/ARTD1) is implicated in the maintenance of silent rDNA chromatin during cell division. We demonstrate that PARP1 associates with TIP5, a subunit of the NoRC complex, via the noncoding pRNA and binds to silent rRNA genes after their replication in mid-late S phase. PARP1 represses rRNA transcription and is implicated in the formation of silent rDNA chromatin. Silent rDNA chromatin is a specific substrate for ADP-ribosylation and the enzymatic activity of PARP1 is necessary to establish rDNA silencing. The data unravel a function of PARP1 and ADP-ribosylation that serves to allow for the inheritance of silent chromatin structures, shedding light on how epigenetic marks are transmitted during each cell cycle. PMID:22405650

  8. Fluorescent DNA-Protected Silver Nanoclusters for Ligand-HIV RNA Interaction Assay.

    PubMed

    Qi, Liang; Huo, Yuan; Wang, Huan; Zhang, Jing; Dang, Fu-Quan; Zhang, Zhi-Qi

    2015-11-01

    Studying ligand-biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) were used to investigate ligand-RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus trans-activation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX-RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand-RNA binding interaction assay. PMID:26447651

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  11. 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 MgCl(2) 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

  12. 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

  13. Flexibility of nucleic acids: from DNA to RNA

    E-print Network

    Lei Bao; Xi Zhang; Lei Jin; Zhi-Jie Tan

    2015-09-23

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids.

  14. Flexibility of nucleic acids: from DNA to RNA

    E-print Network

    Bao, Lei; Jin, Lei; Tan, Zhi-Jie

    2015-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids.

  15. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  17. Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase.

    PubMed Central

    Gadeau, A P; Mouches, C; Bove, J M

    1986-01-01

    The effect of rifampin on five mollicutes (Spiroplasma citri, Spiroplasma melliferum, Spiroplasma apis, Acholeplasma laidlawii, and Mycoplasma mycoides) was compared with that on Escherichia coli. We found that, in contrast to wild-type E. coli, mollicutes were insensitive to rifampin. DNA-dependent RNA polymerases from S. melliferum and S. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. The enzymes were then tested for their sensitivity to rifampin. Spiroplasmal enzymes were at least 1,000 times less sensitive to rifampin than the corresponding E. coli enzyme. This result provides a molecular basis for the resistance of mollicutes to rifampin. The RNA polymerase of S. melliferum was further purified and its subunit composition was investigated. The RNA polymerase has one small and two large subunits. The structure of S. melliferum RNA polymerase therefore resembles that of the eubacterial enzymes in spite of its insensitivity to rifampin. Images PMID:3519581

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

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

    2002-04-01

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

  20. Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids.

    PubMed

    Salvi, Jayesh S; Chan, Janet N Y; Szafranski, Kirk; Liu, Tony T; Wu, Jane D; Olsen, Jonathan B; Khanam, Nurussaba; Poon, Betty P K; Emili, Andrew; Mekhail, Karim

    2014-07-28

    Intergenic transcription within repetitive loci such as the ribosomal DNA (rDNA) repeats of yeast commonly triggers aberrant recombination. Major mechanisms suppressing aberrant rDNA recombination rely on chromatin silencing or RNAPII repression at intergenic spacers within the repeats. We find ancient processes operating at rDNA intergenic spacers and other loci to maintain genome stability via repression of RNA-DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds noncoding RNA, suppresses RNA-DNA hybrids, and prevents aberrant rDNA recombination. Repression of RNA-DNA hybrids in Pbp1-deficient cells through RNaseH overexpression, deletion of the G4DNA-stabilizing Stm1, or caloric restriction operating via RNaseH/Pif1 restores rDNA stability. Pbp1 also limits hybrids at non-rDNA G4DNA loci including telomeres. Moreover, cells lacking Pbp1 have a short replicative lifespan that is extended upon hybrid suppression. Thus, we find roles for Pbp1 in genome maintenance and reveal that caloric restriction counteracts Pbp1 deficiencies by engaging RNaseH and Pif1. PMID:25073155

  1. 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

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

    EPA Science Inventory

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

  3. Recognition of DNA/RNA bulges by antimicrobial and antitumor metallohelices.

    PubMed

    Malina, Jaroslav; Scott, Peter; Brabec, Viktor

    2015-09-01

    Bulged structures have been identified in nucleic acids and have been shown to be linked to biomolecular processes involved in numerous diseases. Thus, chemical agents with affinity for bulged nucleic acids are of general biological significance. Herein, the mechanism of specific recognition and stabilization of bulged DNA and RNA by helical bimetallic species was established through detailed molecular biophysics and biochemistry assays. These agents, known as 'flexicates', are potential mimetics of ?-helical peptides in cancer treatment, exhibiting antimicrobial and antitumor effects. The flexicates have positive impacts on the thermal stability of DNA duplexes containing bulges, which means that the flexicates interact with the duplexes containing bulges, and that these interactions stabilize the secondary structures of these duplexes. Notably, the stabilising effect of the flexicates increases with the size of the bulge, the maximal stabilization is observed for the duplexes containing a bulge composed of at least three bases. The flexicates bind most preferentially to the bulges composed of pyrimidines flanked on both sides also by pyrimidines. It is suggested that it is so because these bulges exhibit greatest conformational variability in comparison with other combinations of bases in the bulge loop and bases flanking the bulge. Finally, the results indicate that there is only one dominant binding site for the flexicates on the DNA and RNA bulges and that the flexicates bind directly to the bulge or in its close proximity. It is also shown that the flexicates effectively bind to RNA duplexes containing the bulged region of HIV-1 TAR RNA. PMID:26212708

  4. Junk DNA and the long non-coding RNA twist in cancer genetics.

    PubMed

    Ling, H; Vincent, K; Pichler, M; Fodde, R; Berindan-Neagoe, I; Slack, F J; Calin, G A

    2015-09-24

    The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single-nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual's susceptibility to cancer. PMID:25619839

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

    PubMed Central

    Yamada, T; Shimaji, M

    1986-01-01

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

  6. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

    PubMed Central

    Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A.; Weinert, Brian T.; Olsen, Jesper V.; Baskcomb, Linda; Mann, Matthias; Jackson, Stephen P.; Choudhary, Chunaram

    2013-01-01

    Summary The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA damaging agents, thus suggesting an important link between RNA metabolism and DNA repair. Our results broaden the knowledge of DNA damage signaling networks and identify novel components of the DDR. PMID:22424773

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

    PubMed

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

    2014-10-01

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

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

    SciTech Connect

    Reich, Charles F.; Pisetsky, David S.

    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.

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

    PubMed Central

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

    1994-01-01

    Xenopus UBF (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. These include a short basic region adjacent to a dimer motif plus five high-mobility-group (HMG) boxes. All of these DNA-binding motifs exhibit low sequence specificity, whether assayed singly or together. In contrast, the HMG boxes recognize DNA structure that is formed when two double helices are crossed over each other. HMG box 1, in particular, requires association of two double helices before it will bind and, either by itself or in the context of the intact protein, will loop DNA and organize it into higher-order structures. We discuss how this mode of binding affects the function of xUBF as a transcription factor. Images PMID:8164649

  10. Blind predictions of DNA and RNA tweezers experiments with force and torque.

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  12. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

    PubMed Central

    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

  13. 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

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

    SciTech Connect

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

    2008-01-01

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

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

    SciTech Connect

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

    2009-11-20

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

  16. 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

  17. 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.

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

    SciTech Connect

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

    1985-07-01

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

  19. 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

  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. 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.

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

    PubMed

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

    2013-02-01

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

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

    SciTech Connect

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

    2009-01-01

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

  4. 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.

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

    PubMed Central

    Andrade, Bruno S; Taranto, Alex G; Góes-Neto, Aristóteles; Duarte, Angelo A

    2009-01-01

    Background The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation. Conclusion This work contributes to the development of new alternatives for controlling the fungal agent of witches' broom disease. PMID:19744344

  7. 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

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

    PubMed Central

    Pan, Feng; Roland, Christopher; Sagui, Celeste

    2014-01-01

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

  9. RNA exosome regulates AID DNA mutator activity in the B cell genome

    PubMed Central

    Pefanis, Evangelos; Basu, Uttiya

    2015-01-01

    The immunoglobulin diversification processes of somatic hypermutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome-wide studies have uncovered pervasive AID activity throughout the B cell genome. It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co-transcriptional RNA degradation mechanisms. Here we review aspects and consequences of eukaryotic transcription that lead to early termination, RNA exosome recruitment, and ultimately targeting of AID mutagenic activity. PMID:26073986

  10. RNA polymerase motor on DNA track: effects of interactions, external force and torque

    E-print Network

    Tripti Tripathi; Prasanjit Prakash; Debashish Chowdhury

    2009-06-04

    RNA polymerase (RNAP) is a mobile molecular workshop that polymerizes a RNA molecule by adding monomeric subunits one by one, while moving step by step on the DNA template itself. Here we develop a theoretical model by incorporating the steric interactions of the RNAPs and their mechanochemical cycles which explicitly captures the cyclical shape changes of each motor. Using this model, we explain not only the dependence of the average velocity of a RNAP on the externally applied load force, but also predict a {\\it nonmotonic} variation of the average velocity on external torque. We also show the effect of steric interactions of the motors on the total rate of RNA synthesis. In principle, our predictions can be tested by carrying out {\\it in-vitro} experiments which we suggest here.

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

    SciTech Connect

    Dugan, L

    2006-02-08

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

  12. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

    PubMed

    Chiappinelli, Katherine B; Strissel, Pamela L; Desrichard, Alexis; Li, Huili; Henke, Christine; Akman, Benjamin; Hein, Alexander; Rote, Neal S; Cope, Leslie M; Snyder, Alexandra; Makarov, Vladimir; Buhu, Sadna; Slamon, Dennis J; Wolchok, Jedd D; Pardoll, Drew M; Beckmann, Matthias W; Zahnow, Cynthia A; Mergoub, Taha; Chan, Timothy A; Baylin, Stephen B; Strick, Reiner

    2015-08-27

    We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model. PMID:26317466

  13. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning

    SciTech Connect

    Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D. )

    1990-10-01

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

  14. The classification of tymoviruses by cDNA-RNA hybridization and other measures of relatedness.

    PubMed

    Blok, J; Gibbs, A; Mackenzie, A

    1987-01-01

    The relationships of twelve tymoviruses have been assessed by cDNA-RNA hybridization. In addition, the percentage molar nucleotide composition of the genome of the PD strain of Kennedya yellow mosaic virus and the percentage molar amino acid composition of the coat proteins of cacao yellow mosaic, Kennedya yellow mosaic and turnip yellow mosaic (Cardamine strain) viruses were estimated. These as well as published serological comparisons and genome and coat protein composition determinations were used to compute classifications of tymoviruses using various "metrics", and simple numerical methods were used to compare the classifications. Measures of relatedness estimated from cDNA-RNA hybridization and base ratio data correlated significantly with each other, but were less closely correlated with those calculated from amino acid data, and did not correlate with those calculated from serological tests. The serological relationships correlated significantly with estimates of relatedness calculated from amino acid data, but not with those based on hybridization or base ratio data. The differences between these classifications mostly resulted from the anomalous behaviour of eggplant mosaic virus, its particles are serologically close to those of other tymoviruses that naturally infect species of the tobacco family, whereas in cDNA-RNA hybridization tests eggplant mosaic virus is closest to the tymoviruses that infect legumes. Similar but smaller anomalies in the characteristics of other tymoviruses were also found. PMID:3662825

  15. 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

  16. H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase.

    PubMed

    Zhou, Jichun; Yang, Lihua; Zhong, Tianyu; Mueller, Martin; Men, Yi; Zhang, Na; Xie, Juanke; Giang, Karolyn; Chung, Hunter; Sun, Xueguang; Lu, Lingeng; Carmichael, Gordon G; Taylor, Hugh S; Huang, Yingqun

    2015-01-01

    DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components. PMID:26687445

  17. SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content.

    PubMed

    Schoft, Vera K; Chumak, Nina; Bindics, János; Slusarz, Lucyna; Twell, David; Köhler, Claudia; Tamaru, Hisashi

    2015-03-01

    Key message: Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify SYBR Green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to SYBR Green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA. PMID:25676347

  18. 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

  19. Primed abortive initiation of RNA synthesis by E. coli RNA polymerase on T7 DNA. Steady state kinetic studies.

    PubMed Central

    Smagowicz, W J; Scheit, K H

    1978-01-01

    Ternary complexes of T7 DNA, RNA polymerase and the antibiotic rifampicin carry out the promoter specific abortive initiation when dinucleoside monophosphates were employed as primers. Primed abortive initiation, leading to synthesis of trinucleoside diphosphates, only occured with combinations of primers and substrates complementary to a promoter region of 8 base pairs centered around the origin of transcription. The steady state kinetics of three abortive initiations at T7 promoter A3 were studied in detail. The reactions appeared to be truly ordered. Affinity constants, maximal velocities and elementary step rate constants were thus obtained. The stimulation by dinucleoside monophosphate primers is brought about by positively effecting the function of the substrate site rather then by their higher affinity to the primer site of the transcriptional complex. PMID:353734

  20. Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

    E-print Network

    Patil, Ashish

    S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in ...

  1. Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs.

    PubMed Central

    Nolan, L L; Fehr, T F

    1987-01-01

    A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined. PMID:3435122

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

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

    SciTech Connect

    Laval, F. )

    1991-05-15

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

  4. Ocular delivery of macromolecules

    PubMed Central

    Kim, Yoo-Chun; Chiang, Bryce; Wu, Xianggen; Prausnitz, Mark R.

    2014-01-01

    Biopharmaceuticals are making increasing impact on medicine, including treatment of indications in the eye. Macromolecular drugs are typically given by physician-administered invasive delivery methods, because non--invasive ocular delivery methods, such as eye drops, and systemic delivery, have low bioavailability and/or poor ocular targeting. There is a need to improve delivery of biopharmaceuticals to enable less-invasive delivery routes, less-frequent dosing through controlled-release drug delivery and improved drug targeting within the eye to increase efficacy and reduce side effects. This review discusses the barriers to drug delivery via various ophthalmic routes of administration in the context of macromolecule delivery and discusses efforts to develop controlled-release systems for delivery of biopharmaceuticals to the eye. The growing number of macromolecular therapies in the eye needs improved drug delivery methods that increase drug efficacy, safety and patient compliance. PMID:24998941

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

    NASA Astrophysics Data System (ADS)

    Kulic, Igor; Nelson, Phil

    2006-03-01

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

  6. Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

    PubMed

    Glassing, Angela; Dowd, Scot E; Galandiuk, Susan; Davis, Brian; Jorden, Jeffrey R; Chiodini, Rodrick J

    2015-12-01

    Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass. PMID:26569458

  7. 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

  8. 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

  9. Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor

    PubMed Central

    Bolduc, Jill M.; Spiegel, P. Clint; Chatterjee, Piyali; Brady, Kristina L.; Downing, Maureen E.; Caprara, Mark G.; Waring, Richard B.; Stoddard, Barry L.

    2003-01-01

    We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 Å resolution. The structure demonstrates the remarkable structural conservation of the ?-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage. PMID:14633971

  10. Selective staining by 4', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

    PubMed

    Kapu?ci?ski, J; Yanagi, K

    1979-08-10

    4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel. PMID:493114

  11. 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

  12. Inhomogeneities and nonlinear dynamics of a helical DNA interacting with a RNA-polymerase

    NASA Astrophysics Data System (ADS)

    Saha, Mirabeau; Kofané, Timoléon C.

    2014-08-01

    We have numerically investigated the effects of helicity and inhomogeneities on DNA base pairs opening. The inhomogeneities are due to the site-dependent stacking and hydrogen bonding energies in DNA and protein molecules. We have considered a situation in which the active site of the RNA-polymerase molecule binds onto the promoter site of the spin-like model of the DNA molecule at the physiological temperature and creates a bubble. During the study, we have found that the helical coupling has to be very weak compared to intra-strand coupling in the real DNA molecule. Results show that inhomogeneities do not affect the general pattern of base pair opening, even as the periodic inhomogeneity introduces a train of periodic oscillations on the tail of the bubble; while the height of the bubble is an increasing function of the helical coupling parameter. The basic properties of breather-like modes, obtained here by taking into account helical structure and inhomogeneities, are essential for DNA functioning since such breathing-like modes are considered to be much better candidates for the nonlinear modes responsible for a locally open state where biological functioning takes place.

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

    SciTech Connect

    Lin, Y.H.; Keil, R.L. )

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

    NASA Astrophysics Data System (ADS)

    Carlon, Enrico; Heim, Thomas

    2006-04-01

    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 good agreement with Langmuir adsorption theory, provided one takes into account the decrease of the effective target concentration due to target-target hybridization in solution. We give an example of a deviation from the expected thermodynamical behavior for the probe set 1091_at due to annotation problems, i.e., the surface-bound probe is not the exact complement of the target RNA sequence, because of errors present in public databases at the time when the array was designed. We show that the parametrization of the experimental data with RNA/DNA free energy improves the quality of the fits and enhances the stability of the fitting parameters compared to previous studies.

  15. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

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

    2013-01-15

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2006-03-21

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

  18. 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.

  19. 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. PMID:26150827

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

    PubMed Central

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

    2014-01-01

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

  1. Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations

    PubMed Central

    Williams, Zev; Ben-Dov, Iddo Z.; Elias, Rony; Mihailovic, Aleksandra; Brown, Miguel; Rosenwaks, Zev; Tuschl, Thomas

    2013-01-01

    We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers. PMID:23440203

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  3. 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

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

    PubMed

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

    2014-08-01

    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

  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. Contact Kinetics in Fractal Macromolecules

    E-print Network

    Dolgushev, Maxim; Blumen, Alexander; Bénichou, Olivier; Voituriez, Raphaël

    2015-01-01

    We consider the kinetics of first contact between two monomers of the same macromolecule. Relying on a fractal description of the macromolecule, we develop an analytical method to compute the Mean First Contact Time (MFCT) for various molecular sizes. In our theoretical description, the non-Markovian feature of monomer motion, arising from the interactions with the other monomers, is captured by accounting for the non-equilibrium conformations of the macromolecule at the very instant of first contact. This analysis reveals a simple scaling relation for the MFCT between two monomers, which involves only their equilibrium distance and the spectral dimension of the macromolecule, independently of its microscopic details. Our theoretical predictions are in excellent agreement with numerical stochastic simulations.

  7. Contact Kinetics in Fractal Macromolecules

    NASA Astrophysics Data System (ADS)

    Dolgushev, Maxim; Guérin, Thomas; Blumen, Alexander; Bénichou, Olivier; Voituriez, Raphaël

    2015-11-01

    We consider the kinetics of first contact between two monomers of the same macromolecule. Relying on a fractal description of the macromolecule, we develop an analytical method to compute the mean first contact time for various molecular sizes. In our theoretical description, the non-Markovian feature of monomer motion, arising from the interactions with the other monomers, is captured by accounting for the nonequilibrium conformations of the macromolecule at the very instant of first contact. This analysis reveals a simple scaling relation for the mean first contact time between two monomers, which involves only their equilibrium distance and the spectral dimension of the macromolecule, independently of its microscopic details. Our theoretical predictions are in excellent agreement with numerical stochastic simulations.

  8. Contact Kinetics in Fractal Macromolecules.

    PubMed

    Dolgushev, Maxim; Guérin, Thomas; Blumen, Alexander; Bénichou, Olivier; Voituriez, Raphaël

    2015-11-13

    We consider the kinetics of first contact between two monomers of the same macromolecule. Relying on a fractal description of the macromolecule, we develop an analytical method to compute the mean first contact time for various molecular sizes. In our theoretical description, the non-Markovian feature of monomer motion, arising from the interactions with the other monomers, is captured by accounting for the nonequilibrium conformations of the macromolecule at the very instant of first contact. This analysis reveals a simple scaling relation for the mean first contact time between two monomers, which involves only their equilibrium distance and the spectral dimension of the macromolecule, independently of its microscopic details. Our theoretical predictions are in excellent agreement with numerical stochastic simulations. PMID:26613478

  9. Generating a long DNA fragment of the target ncRNA for quantitative polymerase chain reaction by combining ncRNA-oligos hybridization and oligos ligation.

    PubMed

    Zeng, Zhen; Zhou, Zheng; Wang, Dan; Wang, Zitian; Yang, Huali; Luo, Jianjun; Chen, Run-Sheng

    2016-01-10

    The poor reproducibility of the reverse transcription combined with quantitative polymerase chain reaction (RT-qPCR) results in an unacceptable reliability of publications based on these data. We established a novel method, in which two short complementary DNA oligos were hybridized with target ncRNA molecules and linked by DNA ligase to obtain a long DNA strand (HL-DNA) replacing cDNA for qPCR detection (HL-qPCR). A series of diluted samples prepared from the same total RNA resource were measured by HL-qPCR and RT-qPCR respectively to acquire their relative concentration of RNU4-1, AK026510 and SNORA73B. For every tested sample, the relative concentration of RNU4-1, AK026510 and SNORA73B obtained by HL-qPCR instead of RT-qPCR is closer to its corresponding true value without significant difference, demonstrating that HL-qPCR exhibits higher accuracy compared with RT-qPCR. With three independent repeats, no significant difference was observed among AK026510/RNU4-1 values of four samples diluted from the same RNA resource, by employing HL-qPCR but not RT-qPCR. It strongly suggests that the good reproducibility of HL-qPCR results from the stable efficiency of HL-DNA production regardless of the concentration and individual features of ncRNA. The novel HL-qPCR could be applied for the regular relative ncRNA concentration detection in the future. PMID:26593981

  10. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    PubMed

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.) [1-2]. In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen for skin and variable results occur for vaginal and nasal mucosa. Lastly, we show that replicates are useful for interpretation of RNA data, as variations can be found even for true technical replicates. Increased numbers of replicates (over four) do, however, not cancel out the impact of this variation on data interpretation. Overall, the results of this study further forensic RNA profiling. PMID:26590860

  11. 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.

  12. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  13. A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange

    PubMed Central

    Zaitsev, Eugene N.; Kowalczykowski, Stephen C.

    2000-01-01

    Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this “inverse” reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein–dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication. PMID:10733533

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

    PubMed

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

    2004-01-12

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

  15. 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…

  16. 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.

  17. RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

    PubMed

    Haas, C; Hanson, E; Banemann, R; Bento, A M; Berti, A; Carracedo, Á; Courts, C; De Cock, G; Drobnic, K; Fleming, R; Franchi, C; Gomes, I; Hadzic, G; Harbison, S A; Hjort, B; Hollard, C; Hoff-Olsen, P; Keyser, C; Kondili, A; Maroñas, O; McCallum, N; Miniati, P; Morling, N; Niederstätter, H; Noël, F; Parson, W; Porto, M J; Roeder, A D; Sauer, E; Schneider, P M; Shanthan, G; Sijen, T; Syndercombe Court, D; Turanská, M; van den Berge, M; Vennemann, M; Vidaki, A; Zatkalíková, L; Ballantyne, J

    2015-05-01

    The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology. PMID:25600397

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

    E-print Network

    Hedges, Blair

    that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes oceanic islands. Despite this ecological diversity and a long evolution- ary history, snakesHigher-Level Snake Phylogeny Inferred from Mitochondrial DNA Sequences of 12s rRNA and 16s r

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    PubMed Central

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

    2013-01-01

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

  1. RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome.

    PubMed

    Li, Qing; Gent, Jonathan I; Zynda, Greg; Song, Jawon; Makarevitch, Irina; Hirsch, Cory D; Hirsch, Candice N; Dawe, R Kelly; Madzima, Thelma F; McGinnis, Karen M; Lisch, Damon; Schmitz, Robert J; Vaughn, Matthew W; Springer, Nathan M

    2015-11-24

    The maize genome is relatively large (?2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (?100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes. PMID:26553984

  2. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    PubMed Central

    Halaby, Marie-Jo; Li, Yan; Harris, Benjamin R.; Jiang, Shuxia; Miskimins, W. Keith; Cleary, Margot P.; Yang, Da-Qing

    2015-01-01

    Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5?-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress. PMID:26273641

  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. Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis

    PubMed Central

    Liebl, Korbinian; Drsata, Tomas; Lankas, Filip; Lipfert, Jan; Zacharias, Martin

    2015-01-01

    Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over- or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure. PMID:26464435

  5. Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis.

    PubMed

    Liebl, Korbinian; Drsata, Tomas; Lankas, Filip; Lipfert, Jan; Zacharias, Martin

    2015-12-01

    Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over- or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure. PMID:26464435

  6. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

    PubMed Central

    Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

    2015-01-01

    CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

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

    NASA Technical Reports Server (NTRS)

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

    2011-01-01

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

  8. Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts

    PubMed Central

    Trost, Brett; Moir, Catherine A.; Gillespie, Zoe E.; Kusalik, Anthony; Mitchell, Jennifer A.; Eskiw, Christopher H.

    2015-01-01

    DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data. PMID:26473061

  9. Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts.

    PubMed

    Trost, Brett; Moir, Catherine A; Gillespie, Zoe E; Kusalik, Anthony; Mitchell, Jennifer A; Eskiw, Christopher H

    2015-09-01

    DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data. PMID:26473061

  10. Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes

    PubMed Central

    Bergues-Pupo, Ana Elisa; Arias-Gonzalez, J. Ricardo; Morón, María Carmen; Fiasconaro, Alessandro; Falo, Fernando

    2015-01-01

    Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\rm {K}^+$\\end{document} central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery. PMID:26170233

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

    PubMed Central

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

    2014-01-01

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

  12. Nonparametric testing for DNA copy number induced differential mRNA gene expression.

    PubMed

    van Wieringen, Wessel N; van de Wiel, Mark A

    2009-03-01

    The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer. We develop nonparametric tests for the detection of copy number induced differential gene expression. The tests incorporate the uncertainty of the calling of genomic aberrations. The test is preceded by a "tuning algorithm" that discards certain genes to improve the overall power of the false discovery rate selection procedure. Moreover, the test statistics are "shrunken" to borrow information across neighboring genes that share the same array CGH signature. For each gene we also estimate its effect, its amount of differential expression due to copy number changes, and calculate the coefficient of determination. The method is illustrated on breast cancer data, in which it confirms previously reported findings, now with a more profound statistical underpinning. PMID:18479479

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    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 valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

  14. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  15. 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

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

    NASA Astrophysics Data System (ADS)

    Liao, Yuhui; Zhou, Xiaoming

    2014-09-01

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

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

    PubMed

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

    2014-07-01

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

  18. A method of precise mRNA/DNA homology-based gene structure prediction

    PubMed Central

    Churbanov, Alexander; Pauley, Mark; Quest, Daniel; Ali, Hesham

    2005-01-01

    Background Accurate and automatic gene finding and structural prediction is a common problem in bioinformatics, and applications need to be capable of handling non-canonical splice sites, micro-exons and partial gene structure predictions that span across several genomic clones. Results We present a mRNA/DNA homology based gene structure prediction tool, GIGOgene. We use a new affine gap penalty splice-enhanced global alignment algorithm running in linear memory for a high quality annotation of splice sites. Our tool includes a novel algorithm to assemble partial gene structure predictions using interval graphs. GIGOgene exhibited a sensitivity of 99.08% and a specificity of 99.98% on the Genie learning set, and demonstrated a higher quality of gene structural prediction when compared to Sim4, est2genome, Spidey, Galahad and BLAT, including when genes contained micro-exons and non-canonical splice sites. GIGOgene showed an acceptable loss of prediction quality when confronted with a noisy Genie learning set simulating ESTs. Conclusion GIGOgene shows a higher quality of gene structure prediction for mRNA/DNA spliced alignment when compared to other available tools. PMID:16242044

  19. 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 ...

  20. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

    PubMed

    Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

    2015-03-31

    Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' ? 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA. PMID:25775526

  1. G-Quadruplex DNA- and RNA-Specific-Binding Proteins Engineered from the RGG Domain of TLS/FUS.

    PubMed

    Takahama, Kentaro; Miyawaki, Arisa; Shitara, Takumi; Mitsuya, Keita; Morikawa, Masayuki; Hagihara, Masaki; Kino, Katsuhito; Yamamoto, Ayumu; Oyoshi, Takanori

    2015-11-20

    Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation. PMID:26360301

  2. Cardiomyocyte Microvesicles Contain DNA/RNA and Convey Biological Messages to Target Cells

    PubMed Central

    Waldenström, Anders; Gennebäck, Nina; Hellman, Urban; Ronquist, Gunnar

    2012-01-01

    Background Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. Methodology/Principal Findings Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. Conclusions/Significance Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted “cardiosomes”, can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes. PMID:22506041

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

    SciTech Connect

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

    2015-01-20

    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. 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, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

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

    DOE PAGESBeta

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

    2015-01-20

    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 synthesismore »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. 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, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.« less

  5. 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

  6. Probing Anomalous Structural Features in Polypurine Tract-Containing RNA–DNA Hybrids with Neomycin B†

    PubMed Central

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

    2009-01-01

    During (?)-strand DNA synthesis in retroviruses and Saccharomyces cerevisiae LTR retrotransposons, a purine rich region of the RNA template, known as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNA-dependent DNA synthesis. Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include runs of purine ribonucleotides, it has been suggested that these PPTs are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism. Here, we have used the aminoglycoside neomycin B (NB) to examine which structural features of the Ty3 PPT contribute to specific recognition and processing by its cognate RT. Using high-resolution NMR, direct infusion FTICR mass spectrometry, and isothermal titration calorimetry, we show that NB binds preferentially and selectively adjacent to the Ty3 3? PPT–U3 cleavage junction and in an upstream 5? region where the thumb subdomain of Ty3 RT putatively grips the substrate. Regions highlighted by NB on the Ty3 PPT are similar to those previously identified on the HIV-1 PPT sequence that are implicated as contact points for substrate binding by its RT. Our findings thus support the notion that common structural features of lentiviral and LTR-retrotransposon PPTs facilitate the interaction with their cognate RT. PMID:19449839

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

    PubMed Central

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

    1992-01-01

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

  8. Macromolecules Relevant to Stone Formation

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  9. Identification of novel markers in rheumatoid arthritis through integrated analysis of DNA methylation and microRNA expression.

    PubMed

    de la Rica, Lorenzo; Urquiza, José M; Gómez-Cabrero, David; Islam, Abul B M M K; López-Bigas, Nuria; Tegnér, Jesper; Toes, René E M; Ballestar, Esteban

    2013-03-01

    Autoimmune rheumatic diseases are complex disorders, whose etiopathology is attributed to a crosstalk between genetic predisposition and environmental factors. Both variants of autoimmune susceptibility genes and environment are involved in the generation of aberrant epigenetic profiles in a cell-specific manner, which ultimately result in dysregulation of expression. Furthermore, changes in miRNA expression profiles also cause gene dysregulation associated with aberrant phenotypes. In rheumatoid arthritis, several cell types are involved in the destruction of the joints, synovial fibroblasts being among the most important. In this study we performed DNA methylation and miRNA expression screening of a set of rheumatoid arthritis synovial fibroblasts and compared the results with those obtained from osteoarthritis patients with a normal phenotype. DNA methylation screening allowed us to identify changes in novel key target genes like IL6R, CAPN8 and DPP4, as well as several HOX genes. A significant proportion of genes undergoing DNA methylation changes were inversely correlated with expression. miRNA screening revealed the existence of subsets of miRNAs that underwent changes in expression. Integrated analysis highlighted sets of miRNAs that are controlled by DNA methylation, and genes that are regulated by DNA methylation and are targeted by miRNAs with a potential use as clinical markers. Our study enabled the identification of novel dysregulated targets in rheumatoid arthritis synovial fibroblasts and generated a new workflow for the integrated analysis of miRNA and epigenetic control. PMID:23306098

  10. 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.

  11. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus.

    PubMed Central

    Lai, C J; Zhao, B T; Hori, H; Bray, M

    1991-01-01

    Dengue virus is an enveloped positive-strand RNA virus with a genome approximately 11 kilobases in length. The four serotypes of dengue virus are currently the most important members of the flavivirus family in terms of geographical distribution and the incidence of infection in humans. In this communication we describe successful cloning of a stable full-length cDNA copy of dengue type 4 virus that can be used as the template for in vitro transcription of infectious RNA. Evidence is presented that dengue virus recovered from permissive cells transfected with the in vitro RNA transcripts retained a mutation that was engineered into full-length cDNA. The properties of the virus produced by cells transfected with infectious RNA transcripts of dengue cDNA resembled those of the virus from which the cDNA clone was derived. The dengue virus recombinant DNA system should prove helpful in gaining a better understanding of the molecular biology of dengue viruses and should facilitate the development of a safe and effective live vaccine for use in humans. Images PMID:2052593

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

    PubMed

    Heidary, David K; Glazer, Edith C

    2014-03-01

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

  13. 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 ...

  14. Repair of UV induced DNA lesions in ribosomal gene chromatin and the role of "Odd" RNA polymerases (I and III).

    PubMed

    Charton, Romain; Guintini, Laetitia; Peyresaubes, François; Conconi, Antonio

    2015-12-01

    In fast growing eukaryotic cells, a subset of rRNA genes are transcribed at very high rates by RNA polymerase I (RNAPI). Nuclease digestion-assays and psoralen crosslinking have shown that they are open; that is, largely devoid of nucleosomes. In the yeast Saccharomyces cerevisae, nucleotide excision repair (NER) and photolyase remove UV photoproducts faster from open rRNA genes than from closed and nucleosome-loaded inactive rRNA genes. After UV irradiation, rRNA transcription declines because RNAPI halt at UV photoproducts and are then displaced from the transcribed strand. When the DNA lesion is quickly recognized by NER, it is the sub-pathway transcription-coupled TC-NER that removes the UV photoproduct. If dislodged RNAPI are replaced by nucleosomes before NER recognizes the lesion, then it is the sub-pathway global genome GG-NER that removes the UV photoproducts from the transcribed strand. Also, GG-NER maneuvers in the non-transcribed strand of open genes and in both strands of closed rRNA genes. After repair, transcription resumes and elongating RNAPI reopen the rRNA gene. In higher eukaryotes, NER in rRNA genes is inefficient and there is no evidence for TC-NER. Moreover, TC-NER does not occur in RNA polymerase III transcribed genes of both, yeast and human fibroblast. PMID:26411875

  15. Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    PubMed Central

    Pontvianne, Frédéric; Abou-Ellail, Mohamed; Douet, Julien; Comella, Pascale; Matia, Isabel; Chandrasekhara, Chinmayi; DeBures, Anne; Blevins, Todd; Cooke, Richard; Medina, Francisco J.; Tourmente, Sylvette; Pikaard, Craig S.; Sáez-Vásquez, Julio

    2010-01-01

    In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis. PMID:21124873

  16. 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

  17. Sequence requirement for hand-in-hand interaction in formation of RNA dimers and hexamers to gear phi29 DNA translocation motor.

    PubMed Central

    Chen, C; Zhang, C; Guo, P

    1999-01-01

    Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that phi29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23-97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggests that segment 23-97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1-22 and 98-120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization. PMID:10376879

  18. Ultrafast Dynamics in DNA and RNA Derivatives Monitored by Broadband Transient Absorption Spectrscopy

    NASA Astrophysics Data System (ADS)

    Brister, Matthew M.; Crespo-Hernández, Carlos E.

    2015-06-01

    The ultrafast dynamics of nucleic acids have been under scrutiny for the past couple of decades because of the role that the high-energy electronic states play in mutagenesis and carcinogenesis. Kinetic models have been proposed, based on both experimental and theoretical discoveries. Direct experimental evidence of the intersystem crossing rate and population of the triplet state for most nucleic acid bases has yet to be reported, even though the triplet state is thought to be the most reactive species. Utilizing broadband femtosecond transient absorption spectroscopy, we reveal the time scale at which singlet-to-triplet population transfer occurs in several nucleic acid derivatives in the condensed phase. The implication of these results to the current understanding of the DNA and RNA photochemistry will be discussed. The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.

  19. Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes

    NASA Astrophysics Data System (ADS)

    Kimsey, Isaac J.; Petzold, Katja; Sathyamoorthy, Bharathwaj; Stein, Zachary W.; Al-Hashimi, Hashim M.

    2015-03-01

    Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10-3 to 10-5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

  20. Macromolecules in Undergraduate Physical Chemistry.

    ERIC Educational Resources Information Center

    Mattice, Wayne L.

    1981-01-01

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

  1. Ion binding to biological macromolecules

    PubMed Central

    Petukh, Marharyta; Alexov, Emil

    2015-01-01

    Biological macromolecules carry out their functions in water and in the presence of ions. The ions can bind to the macromolecules either specifically or non-specifically, or can simply to be a part of the water phase providing physiological gradient across various membranes. This review outlines the differences between specific and non-specific ion binding in terms of the function and stability of the corresponding macromolecules. Furthermore, the experimental techniques to identify ion positions and computational methods to predict ion binding are reviewed and their advantages compared. It is indicated that specifically bound ions are relatively easier to be revealed while non-specifically associated ions are difficult to predict. In addition, the binding and the residential time of non-specifically bound ions are very much sensitive to the environmental factors in the cells, specifically to the local pH and ion concentration. Since these characteristics differ among the cellular compartments, the non-specific ion binding must be investigated with respect to the sub-cellular localization of the corresponding macromolecule. PMID:25774076

  2. Physical Localization and DNA Methylation of 45S rRNA Gene Loci in Jatropha curcas L.

    PubMed Central

    Gong, Zhiyun; Xue, Chao; Zhang, Mingliang; Guo, Rui; Zhou, Yong; Shi, Guoxin

    2013-01-01

    In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n?=?2x?=?22?=?12m+10sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation. PMID:24386362

  3. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    PubMed Central

    Gammon, Don B; Duraffour, Sophie; Rozelle, Daniel K; Hehnly, Heidi; Sharma, Rita; Sparks, Michael E; West, Cara C; Chen, Ying; Moresco, James J; Andrei, Graciela; Connor, John H; Conte, Darryl; Gundersen-Rindal, Dawn E; Marshall, William L; Yates, John R; Silverman, Neal; Mello, Craig C

    2014-01-01

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-?B, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001 PMID:24966209

  4. 'Effects of Elevated Temperature on Dehalococcoides Dechlorination Performance and DNA and RNA Biomarker Abundance

    SciTech Connect

    Fletcher, Kelly E; Costanza, Jed; Cruz-Garcia, Claribel; Ramaswamy, Nivedhya; Pennell, Kurt; Loeffler, Frank E

    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.

  5. Human apolipoproteins AI, AII, CII and CIII. cDNA sequences and mRNA abundance.

    PubMed Central

    Sharpe, C R; Sidoli, A; Shelley, C S; Lucero, M A; Shoulders, C C; Baralle, F E

    1984-01-01

    The structure and function of the genes encoding the polypeptide components of plasma lipoproteins are of interest because of the central role they play in the regulation of lipid metabolism. We have now completed our previous studies on the human apoAI gene and furthermore isolated and sequenced cDNA clones for apoAII , CII and CIII. The nucleotide sequences show the signal peptides of apoAII , CII and CIII to be 18, 22 and 20 amino acids in length, respectively, and in addition that prepro apoAII bears a classical propeptide structure of 5 amino acids. The amino acid homology detected between apoCII and pro- apoAI is discussed, as is the gene arrangement of the 5' non-coding region of apoAI mRNA. The relative liver mRNA levels of the 4 apolipoproteins analysed in this study have been estimated and compared with their corresponding plasma products. The data reported here provide an essential basis for further studies of structural and functional alleles of apo AI, AII, CII and CIII genes. Images PMID:6328445

  6. 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

  7. 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 of the antimicrobial activ- ity spectrum of A. aegypti and Drosophila cecropin A showed a lower activity production of potent antimicrobial peptides effective against bacteria and fungi; transcriptional activity

  8. A New Three-Dimensional Educational Model Kit for Building DNA and RNA Molecules: Development and Evaluation

    ERIC Educational Resources Information Center

    Beltramini, Leila Maria; Araujo, Ana Paula Ulian; de Oliveira, Tales Henrique Goncalves; dos Santos Abel, Luciano Douglas; da Silva, Aparecido Rodrigues; dos Santos, Neusa Fernandes

    2006-01-01

    International specialized literature focused on research in biology education is sadly scarce, especially regarding biochemical and molecular aspects. In this light, researchers from this Centre for Structural Molecular Biotechnology developed and evaluated a three-dimensional educational model named "Building Life Molecules DNA and RNA." The…

  9. Histidine-rich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway.

    PubMed

    Vu, Trang T; Leslie, Beverly A; Stafford, Alan R; Zhou, Ji; Fredenburgh, James C; Weitz, Jeffrey I

    2015-12-22

    When triggered by factor (F) XII and nucleic acids, we showed that thrombosis in HRG-deficient mice is accelerated compared with that in wild-type mice. In this study, we set out to identify the mechanisms by which nucleic acids promote contact activation, and to determine whether HRG attenuates their effects. DNA or RNA addition to human plasma enhances thrombin generation via the intrinsic pathway and shortens the clotting time. Their effect on the clotting time is seven- to 14-fold greater in HRG-deficient plasma than in control plasma. Investigations into the mechanisms of activation reveal that nucleic acids a) promote FXII activation in the presence of prekallikrein- and high molecular weight kininogen (HK), and b) enhance thrombin-mediated FXI activation by 10- to 12-fold. Surface plasmon resonance studies show that DNA and RNA bind FXII, FXIIa, HK, FXI, FXIa and thrombin with high affinity. HRG attenuates DNA- and RNA-mediated FXII activation, and FXI activation by FXIIa or by thrombin, suggesting that HRG down regulates the capacity of DNA and RNA to activate the intrinsic pathway. Therefore, HRG attenuates the procoagulant activity of nucleic acids at multiple levels. PMID:26354857

  10. Development of a rapid isolation assay of high quality RNA and DNA from several peanut tissues suitable for molecular analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of high quality peanut RNA and DNA is a prerequisite for transcript analyses and genetic studies. The presence of phenolic compounds and polysaccharides in peanut tissue can decrease yield quantity and quality which may render the isolated nucleic acid products unsuitable for various mole...

  11. SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation

    E-print Network

    Jacobsen, Steve

    SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant pro- teins, SNF2-RING-HELICASE­LIKE1 and -2 (FRG1 and -2) that are puta- tive chromatin modifiers belonging to the SNF2

  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. Defining the starvation potential and the influence on RNA/DNA ratios in horse mackerel ( Trachurus mediterraneus) larvae

    NASA Astrophysics Data System (ADS)

    Yandi, Ilhan; Altinok, Ilhan

    2015-03-01

    Larval survival potentially affects recruitment strongly. Variability in larval growth rates, primarily caused by variable nutritional situations, is one of the factors that can influence larval survival rates. RNA/DNA ratio as well as protein content was analyzed in wild-caught laboratory-grown and in wild-caught horse mackerel Trachurus mediterraneus in relation to feeding and starvation. For this purpose, field-caught genoblast eggs were incubated and the hatched larvae were reared under different feeding regimes: fed control, unfed control, starved either for 1, 2 or 3 days, on feeding restrictions. The whole-body RNA/DNA ratio and the daily protein growth rate were individually analyzed. In all larvae eye pigmentation, mouth opening and subsequently first feeding started on the third day after hatching. All larvae in the unfed group died on day 8. The survival rate during the first 3 days in delayed feeding groups was higher than that of the unfed group. Overall, growth curves from feeding-delayed larvae indicated that fish fed after up to 3 days starvation were capable of complete recovery with the critical RNA/DNA ratio of 1.05 ± 0.08. According to this value, approximately 10 % of the field-caught larvae were starving. Therefore, the RNA/DNA ratio is an easy tool to assess the nutritional status in horse mackerel larvae caught in the field with a high precision rate.

  14. 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

  15. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression

    PubMed Central

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker

    2015-01-01

    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system. PMID:26068470

  16. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage

    PubMed Central

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M.; Espinar, Lorena; Blevins, William R.; Lehner, Ben; Carey, Lucas B.

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method—FitFlow—that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic—and so phenotypic — space. PMID:26268986

  17. Conformation and orientation dependence in ion-induced collisions with DNA and RNA building blocks

    NASA Astrophysics Data System (ADS)

    Bacchus-Montabonel, Marie-Christine

    2015-04-01

    Action of radiations on biological tissues is of major concern in cancer therapy development. Understanding the mechanisms involved at the molecular level in such reactions may be of crucial interest. In particular ion-induced ionization processes appear at the early stage of damage and a detailed analysis has been performed on the charge transfer dynamics of carbon ions with the different DNA and RNA building blocks in order to analyze their respective behavior in ion-induced collisions. We have considered the pyrimidine nucleobases uracil and thymine and the 5-halouracil molecules corresponding to the same skeleton, as well as the sugar moiety 2-deoxy-D-ribose. The calculations have been performed by means of ab initio quantum chemistry molecular methods followed by a semi-classical collision treatment in a wide collision energy range. Considerations of the structure of the biological target as well as analysis of the anisotropy of the process have been performed. The comparison with proton collisions has been developed with regard to previous results. Qualitative trends of interest for DNA building blocks damage may be pointed out.

  18. 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.

  19. 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

  20. Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar?dominance

    PubMed Central

    Chen, Z. Jeffrey; Pikaard, Craig S.

    1997-01-01

    Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the ?3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2?-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also de-epressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA. PMID:9284051

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

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

    2014-11-01

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

  3. Regulation of plastid rDNA transcription by interaction of CDF2 with two different RNA polymerases.

    PubMed

    Bligny, M; Courtois, F; Thaminy, S; Chang, C C; Lagrange, T; Baruah-Wolff, J; Stern, D; Lerbs-Mache, S

    2000-04-17

    The plastid genome is known to be transcribed by a plastid-encoded prokaryotic-type RNA polymerase (PEP) and by a nucleus-encoded phage-type RNA polymerase (NEP). The spinach plastid rrn operon promoter region harbours three different, overlapping promoters. Two of them are of the prokaryotic type. The third promoter is a non-consensus-type NEP promoter. We separated three different transcriptional activities from spinach chloroplasts: PEP, the phage-type RNA polymerase NEP-1, and a third, hitherto undescribed transcriptional activity (NEP-2). NEP-2 specifically transcribes the rrn operon in the presence of the transcription factor CDF2. CDF2 was previously shown to recruit PEP to the rrn promoter to repress transcription. Together, our results suggest the existence of a third RNA polymerase in plastids and a mechanism of rDNA transcriptional regulation that is based on the interaction of the transcription factor CDF2 with two different transcriptional systems. PMID:10775269

  4. Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase

    PubMed Central

    Ditlevson, Jennifer V.; Tornaletti, Silvia; Belotserkovskii, Boris P.; Teijeiro, Virginia; Wang, Guliang; Vasquez, Karen M.; Hanawalt, Philip C.

    2008-01-01

    DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)14, which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)14 sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)14 sequence, when the purine–pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)14 sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product. PMID:18400779

  5. Catalytic Activity of a Binary Informational Macromolecule

    NASA Technical Reports Server (NTRS)

    Reader, John S.; Joyce, Gerald F.

    2003-01-01

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

  6. 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

  7. 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

  8. The Epstein-Barr Virus (EBV) SM Protein Enhances Pre-mRNA Processing of the EBV DNA Polymerase Transcript

    PubMed Central

    Key, S. Catherine Silver; Yoshizaki, Tomokazu; Pagano, Joseph S.

    1998-01-01

    The Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA, which contains a noncanonical polyadenylation signal, UAUAAA, is cleaved and polyadenylated inefficiently (S. C. S. Key and J. S. Pagano, Virology 234:147–159, 1997). We postulated that the EBV early proteins SM and M, which appear to act posttranscriptionally and are homologs of herpes simplex virus (HSV) ICP27, might compensate for the inefficient processing of pol pre-mRNA. Here we show that the SM and M proteins interact with each other in vitro. In addition, glutathione S-transferase–SM/M fusion proteins precipitate the heterogeneous ribonucleoprotein (hnRNP) C1 splicing protein. Further, the SM protein is coimmunoprecipitated from SM-expressing cell extracts with an antibody to the hnRNP A1/A2 proteins, which are splicing and nuclear shuttling proteins. Finally, the amount of processed EBV DNA polymerase mRNA was increased three- to fourfold in a HeLa cell line expressing SM; this increase was not due to enhanced transcription. Thus, inefficient processing of EBV pol RNA by cellular cleavage and polyadenylation factors appears to be compensated for and may be regulated by the early EBV protein, SM, perhaps via RNA 3?-end formation. PMID:9765385

  9. 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. PMID:26150987

  10. Molecular Testing for miRNA, mRNA, and DNA on Fine-Needle Aspiration Improves the Preoperative Diagnosis of Thyroid Nodules With Indeterminate Cytology

    PubMed Central

    Shifrin, Alexander; Busseniers, Anne E.; Lupo, Mark A.; Manganelli, Monique L.; Andruss, Bernard; Wylie, Dennis; Beaudenon-Huibregtse, Sylvie

    2015-01-01

    Context: Molecular testing for oncogenic mutations or gene expression in fine-needle aspirations (FNAs) from thyroid nodules with indeterminate cytology identifies a subset of benign or malignant lesions with high predictive value. Objective: This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Setting: Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the miRInform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Design: Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Results: Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73–97%) and 85% (95% CI, 75–92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85–98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. Conclusions: Multiplatform testing for DNA, mRNA, and miRNA can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology. PMID:25965083

  11. Gene Silencing Using 4'-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response.

    PubMed

    Tarashima, Noriko; Ando, Hidenori; Kojima, Takamitsu; Kinjo, Nozomi; Hashimoto, Yosuke; Furukawa, Kazuhiro; Ishida, Tatsuhiro; Minakawa, Noriaki

    2016-01-01

    The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4'-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4'-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4'-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response. PMID:26730811

  12. Micro(mi) RNA-34a targets protein phosphatase (PP)1? to regulate DNA damage tolerance.

    PubMed

    Takeda, Yuko; Venkitaraman, Ashok R

    2015-12-17

    The DNA damage response (DDR) triggers widespread changes in gene expression, mediated partly by alterations in micro(mi) RNA levels, whose nature and significance remain uncertain. Here, we report that miR-34a, which is upregulated during the DDR, modulates the expression of protein phosphatase 1? (PP1?) to regulate cellular tolerance to DNA damage. Multiple bio-informatic algorithms predict that miR-34a targets the PP1CCC gene encoding PP1? protein. Ionising radiation (IR) decreases cellular expression of PP1? in a dose-dependent manner. An miR-34a-mimic reduces cellular PP1? protein. Conversely, an miR-34a inhibitor antagonizes IR-induced decreases in PP1? protein expression. A wild-type (but not mutant) miR-34a seed match sequence from the 3' untranslated region (UTR) of PP1CCC when transplanted to a luciferase reporter gene makes it responsive to an miR-34a-mimic. Thus, miR-34a upregulation during the DDR targets the 3' UTR of PP1CCC to decrease PP1? protein expression. PP1? is known to antagonize DDR signaling via the ataxia-telangiectasia-mutated (ATM) kinase. Interestingly, we find that cells exposed to DNA damage become more sensitive - in an miR-34a-dependent manner - to a second challenge with damage. Increased sensitivity to the second challenge is marked by enhanced phosphorylation of ATM and p53, increased ?H2AX formation, and increased cell death. Increased sensitivity can be partly recapitulated by a miR-34a-mimic, or antagonized by an miR-34a-inhibitor. Thus, our findings suggest a model in which damage-induced miR-34a induction reduces PP1? expression and enhances ATM signaling to decrease tolerance to repeated genotoxic challenges. This mechanism has implications for tumor suppression and the response of cancers to therapeutic radiation. PMID:26111201

  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. Unwinding DNA and RNA with Synthetic Complexes: On the Way to Artificial Helicases.

    PubMed

    Gasiorek, Martin; Schneider, Hans-Jörg

    2015-12-01

    Synthetic helicases can be designed on the basis of ligands that bind more strongly to single-stranded nucleic acids than to double-stranded nucleic acids. This can be achieved with ligands containing phenyl groups, which intercalate into single strands, but due to their small size not into double strands. Moreover, two phenyl rings are combined with a distance that allows bis-intercalation with only single strands and not double strands. In this respect, such ligands also mimic single-strand binding (SSB) proteins. Exploration with more than 23 ligands, mostly newly synthesised, shows that the distance between the phenyl rings and between those and the linker influence the DNA unwinding efficiency, which can reach a melting point decrease of almost ?Tm =50?°C at much lower concentrations than that with any other known artificial helicases. Conformational pre-organisation of the ligand plays a decisive role in optimal efficiency. Substituents at the phenyl rings have a large effect, and increase, for example, in the order of HRNA model one observes unfolding at 29?°C with a concentration of only 30??M. PMID:26503404

  15. Magnesium effect on premelting transitions in nucleic acids: DNA duplex and RNA hairpin models

    NASA Astrophysics Data System (ADS)

    Ottová, Pavla; Espinoza-Herrera, Shirly Josefina; Št?pánek, Josef

    2011-05-01

    The effect of magnesium ions on the conformational changes in the temperature region below the melting transition (premelting transitions) of a DNA duplex and an RNA hairpin was studied by difference Raman spectroscopy. The chosen model systems were the polynucleotide poly(dA)-poly(dT) duplex and the apical hairpin of the trans-activation response (TAR) element of HIV-1. Magnesium effect was displayed by differences of Raman spectra measured at various temperatures with and without magnesium ions. Our results have revealed that magnesium ions influence measurably the premelting transitions in both cases; the extent of the effect and its character is though quite different. In the case of the B' ? B premelting transition of the polynucleotide poly(dA)-poly(dT) duplex, magnesium binds to the minor groove of the B but not of the B' form and acts again the rearrangement in the vicinity of the thymidine keto-groups. On the other hand, the magnesium effect on the TAR hairpin premelting consists in a weak support of the premelting structural change via more effective electrostatic shielding.

  16. Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

    PubMed

    Glushakova, Lyudmyla G; Sharma, Nidhi; Hoshika, Shuichi; Bradley, Andrea C; Bradley, Kevin M; Yang, Zunyi; Benner, Steven A

    2015-11-15

    Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-?l sample. PMID:26299645

  17. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  18. Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor ?Q

    PubMed Central

    Vorobiev, Sergey M.; Gensler, Yocheved; Vahedian-Movahed, Hanif; Seetharaman, Jayaraman; Su, Min; Huang, Janet Y.; Xiao, Rong; Kornhaber, Gregory; Montelione, Gaetano T.; Tong, Liang; Ebright, Richard H.; Nickels, Bryce E.

    2014-01-01

    SUMMARY The bacteriophage ? Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, ?Q binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. ?Q’s interaction with the paused early elongation complex involves interactions between ?Q and two regions of RNA polymerase: region 4 of the ?70 subunit and the flap domain of the ? subunit. We present the 2.1 Å resolution crystal structure of a portion of ?Q containing determinants for interaction with DNA, interaction with region 4 of ?70, and interaction with the ? flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which ?Q alters the functional properties of the transcription elongation complex. PMID:24440517

  19. Differential introduction of DNA damage and repair in mammalian genes transcribed by RNA polymerases I and II.

    PubMed Central

    Vos, J M; Wauthier, E L

    1991-01-01

    We have developed a general quantitative method for comparing the levels of drug-induced DNA crosslinking in specific mammalian genes. We observed a dramatic difference between the efficiency of the removal of both psoralen monoadducts and interstrand crosslinks from the rRNA genes and the efficiency of their removal from the dihydrofolate reductase (DHFR) gene in cultured human and hamster cells. While 90% of the interstand crosslinks were removed from the human DHFR gene in 48 h, less than 25% repair occurred in the rRNA genes. Similarly, in Chinese hamster ovary cells, 85% repair of interstrand crosslinks occurred within 8 h in the DHFR gene versus only 20% repair in the rRNA genes. The preferential repair of the DHFR gene relative to that of the rRNA genes was also observed for psoralen monoadducts in cells from both mammalian species. In human-mouse hybrid cells, the active mouse rRNA genes were five times more susceptible to psoralen modification than are the silent rRNA human genes, but adduct removal was similarly inefficient for both classes. We conclude that the repair of chemical damage such as psoralen photoadducts in an expressed mammalian gene may depend upon the class of transcription to which it belongs. Images PMID:2005908

  20. Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays.

    PubMed

    Yagmur, Gulhan; Altun, Hatice Uludag; Gökahmetoglu, Selma; Basok, Ela

    2015-09-01

    In the diagnosis and monitoring of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, it is important to use methods that can provide rapid and reliable results. The present study aimed to compare the automated and manual extraction methods during the nucleic acid isolation phase for HBV-DNA and HCV-RNA assays. The study included 93 serum samples, 49 of which were for the HBV-DNA assay and 44 for the HCV-RNA assay. DNA and RNA isolation from the samples was performed manually with a "QIAmpMin Elute Kit" (Qiagen, Germany) and the automated isolation system, NucliSens easyMAG (BioMérieux, France). All the extraction products were amplified using the iCycler device (Bio-Rad, USA). With both methods, compliance was found in 21 (42.8%) samples in the HBV-DNA assay; nine (18.3%) samples had a higher amount of viral nucleic acid with the manual method, whereas 19 samples (38.7%) were found to have a higher amount of nucleic acid with the automated system. For the HCV-RNA assay, total compliance was found in 31 (70.4%) samples; 12 (27.2%) samples had a higher amount of viral nucleic acid with the manual method whereas one sample (2.2%) was found to have a higher amount of nucleic acid with the automated system. It was concluded that the NucliSens easyMAG automated isolation system can be used with confidence for nucleic acid extraction due to its higher sensitivity, providing results in a shorter time, and assured standardization. PMID:26397294

  1. Phylogenetic analysis of DNA and RNA polymerases from a Moniliophthora perniciosa mitochondrial plasmid reveals probable lateral gene transfer.

    PubMed

    Andrade, B S; Góes-Neto, A

    2015-01-01

    The filamentous fungus Moniliophthora perniciosa is a hemibiotrophic basidiomycete that causes witches' broom disease of cacao (Theobroma cacao L.). Many fungal mitochondrial plasmids are DNA and RNA polymerase-encoding invertrons with terminal inverted repeats and 5'-linked proteins. The aim of this study was to carry out comparative and phylogenetic analyses of DNA and RNA polymerases for all known linear mitochondrial plasmids in fungi. We performed these analyses at both gene and protein levels and assessed differences between fungal and viral polymerases in order to test the lateral gene transfer (LGT) hypothesis. We analyzed all mitochondrial plasmids of the invertron type within the fungal clade, including five from Ascomycota, seven from Basidiomycota, and one from Chytridiomycota. All phylogenetic analyses generated similar tree topologies regardless of the methods and datasets used. It is likely that DNA and RNA polymerase genes were inserted into the mitochondrial genomes of the 13 fungal species examined in our study as a result of different LGT events. These findings are important for a better understanding of the evolutionary relationships between fungal mitochondrial plasmids. PMID:26535725

  2. Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands

    PubMed Central

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

    2008-01-01

    The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5?(rA)4:(dT)4 tract and the 3? PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure. PMID:18400780

  3. Inhibition of DNA nanotube-conjugated mTOR siRNA on the growth of pulmonary arterial smooth muscle cells.

    PubMed

    You, Zaichun; Qian, Hang; Wang, Changzheng; He, Binfeng; Yan, Jiawei; Mao, Chengde; Wang, Guansong

    2015-12-01

    Here we provide raw and processed data and methods behind mTOR siRNA loaded DNA nanotubes (siRNA-DNA-NTs) in the growth of pulmonary arterial smooth muscle cells (PASMCs) under both normoxic and hypoxic condition, and also related to (You et al., Biomaterials, 2015, 67:137-150, [1]). The MTT analysis, Semi-quantitative RT-PCR data presented here were used to probe cytotoxicity of mTOR siRNA-DNA-NT complex in its TAE-Mg(2+) buffer. siRNA-DNA-NTs have a lower cytotoxicity and higher transfection efficiency and can, based on inhibition of mTOR expression, decrease PASMCs growth both hypoxic and normal condition. PMID:26380842

  4. 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...

  5. 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 ...

  6. Reversal of P-glycoprotein-mediated multidrug resistance by CD44 antibody-targeted nanocomplexes for short hairpin RNA-encoding plasmid DNA delivery.

    PubMed

    Gu, Jijin; Fang, Xiaoling; Hao, Junguo; Sha, Xianyi

    2015-03-01

    Multidrug resistance (MDR) remains one of the major reasons for the reductions in efficacy of many chemotherapeutic agents in cancer therapy. As a classical MDR phenotype of human malignancies, the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp) is an efflux protein with aberrant activity that has been linked to multidrug resistance in cancer. For the reversal of MDR by RNA interference (RNAi) technology, an U6-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed (abbreviated pDNA-iMDR1-shRNA). This study explored the feasibility of using Pluronic P123-conjugated polypropylenimine (PPI) dendrimer (P123-PPI) as a carrier for pDNA-iMDR1-shRNA to overcome tumor drug resistance in breast cancer cells. P123-PPI functionalized with anti-CD44 monoclonal antibody (CD44 receptor targeting ligand) (anti-CD44-P123-PPI) can efficiently condense pDNA into nanocomplexes to achieve efficient delivery of pDNA, tumor specificity and long circulation. The in vitro studies methodically evaluated the effect of P123-PPI and anti-CD44-P123-PPI on pDNA-iMDR1-shRNA delivery and P-gp downregulation. Our in vitro results indicated that the P123-PPI/pDNA and anti-CD44-P123-PPI/pDNA nanocomplexes with low cytotoxicity revealed higher transfection efficiency compared with the PPI/pDNA nanocomplexes and Lipofectamine™ 2000 in the presence of serum. The nanocomplexes loaded with pDNA-iMDR1-shRNA against P-gp could reverse MDR accompanied by the suppression of MDR1/P-gp expression at the mRNA and protein levels and improve the internalization and cytotoxicity of Adriamycin (ADR) in the MCF-7/ADR multidrug-resistant cell line. BALB/c nude mice bearing MCF-7/ADR tumor were utilized as a xenograft model to assess antitumor efficacy in vivo. The results demonstrated that the administration of anti-CD44-P123-PPI/pDNA-iMDR1-shRNA nanocomplexes combined with ADR could inhibit tumor growth more efficiently than ADR alone. The enhanced therapeutic efficacy of ADR may be correlated with increased accumulation of ADR in drug-resistant tumor cells. Consequently, these results suggested that the use of pDNA-iMDR1-shRNA-loaded nanocomplexes may be a promising gene delivery strategy to reverse MDR and improve the effectiveness of chemotherapy. PMID:25662500

  7. 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

  8. The introduction of RNA-DNA differences underlies interindividual variation in the human IL12RB1 mRNA repertoire.

    PubMed

    Turner, Amy J; Aggarwal, Praful; Miller, Halli E; Waukau, Jill; Routes, John M; Broeckel, Ulrich; Robinson, Richard T

    2015-12-15

    Human interleukin 12 and interleukin 23 (IL12/23) influence susceptibility or resistance to multiple diseases. However, the reasons underlying individual differences in IL12/23 sensitivity remain poorly understood. Here we report that in human peripheral blood mononuclear cells (PBMCs) and inflamed lungs, the majority of interleukin-12 receptor ?1 (IL12RB1) mRNAs contain a number of RNA-DNA differences (RDDs) that concentrate in sequences essential to IL12R?1's binding of IL12p40, the protein subunit common to both IL-12 and IL-23. IL12RB1 RDDs comprise multiple RDD types and are detectable by next-generation sequencing and classic Sanger sequencing. As a consequence of these RDDs, the resulting IL12R?1 proteins have an altered amino acid sequence that could not be predicted on the basis of genomic DNA sequencing alone. Importantly, the introduction of RDDs into IL12RB1 mRNAs negatively regulates IL12R?1's binding of IL12p40 and is sensitive to activation. Collectively, these results suggest that the introduction of RDDs into an individual's IL12RB1 mRNA repertoire is a novel determinant of IL12/23 sensitivity. PMID:26621740

  9. 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

  10. A primer-free method that selects high affinity single stranded DNA aptamers using Thermostable RNA Ligase

    PubMed Central

    Lai, Yi-Tak; DeStefano, Jeffrey J.

    2011-01-01

    This report describes a method for selecting single stranded DNA (ssDNA) molecules that bind with high affinity (aptamers) to specific target proteins. This SELEX (Systematic Evolution of Ligands by EXponential enrichment) method is similar to other “primer-free” approaches where the random sequence ssDNA starting pool has no fixed sequences at the 5? and 3? termini. Therefore there are no predetermined sequences that could bias selection. Like other SELEX methods, repeated cycles (typically 5–15) of selection then amplification and re-selection are used. The method differs from other primer-free approaches in that the key step for regenerating new material for subsequent rounds is ligation of the selected ssDNA to a defined sequence oligonucleotide using Thermostable RNA Ligase. Under specific conditions, this ligase ligated 30 nucleotide random sequence ssDNA (5?-N30-3?) to a specified 20 nucleotide ssDNA with ~50% efficiency. Efficiency was improved to ~90% by addition of a single T residue to the 3? end (5?-N29T-3?). High efficiency in this step is critical, especially early in the procedure as any selected material that is not ligated is lost. In this report, human immunodeficiency virus reverse transcriptase was used as the target protein but the method could be applied to essentially any protein. PMID:21420926

  11. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

    SciTech Connect

    Jones, E.V.; Moss, B.

    1984-01-01

    The previous demonstration that a phosphonoacetate (PAA)-resistant (PAA/sup r/) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAA/sup s/) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAA/sup r/ mutant virus. Formation of PAA/sup r/ recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAA/sup r/ phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAA/sup r/ vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

  12. Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation[W][OPEN

    PubMed Central

    Gent, Jonathan I.; Madzima, Thelma F.; Bader, Rechien; Kent, Matthew R.; Zhang, Xiaoyu; Stam, Maike; McGinnis, Karen M.; Dawe, R. Kelly

    2014-01-01

    RNA-directed DNA methylation (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2. The data revealed that the majority of the genome exists in a heterochromatic state defined by inaccessible chromatin that is marked by H3K9me2 and H3K27me2 but that lacks RdDM. The minority of the genome marked by RdDM was predominantly near genes, and its overall chromatin structure appeared more similar to euchromatin than to heterochromatin. These and other data indicate that the densely staining chromatin defined as heterochromatin differs fundamentally from RdDM-targeted chromatin. We propose that small interfering RNAs perform a specialized role in repressing transposons in accessible chromatin environments and that the bulk of heterochromatin is incompatible with small RNA production. PMID:25465407

  13. 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.

  14. Configurational diffusion of coal macromolecules

    SciTech Connect

    Guin, J.A.; Curtis, C.W.; Tarrer, A.R.; Kim, S.; Hwang, D.; Chen, C.C.; Chiou, Z.

    1991-01-01

    The objective of our research was to obtain fundamental information regarding the functional dependence of the diffusion coefficient of coal molecules on the ratio of molecule to pore diameter. That is, the objective of our study was to examine the effect of molecule size and configuration on hindered diffusion of coal macromolecules through as porous medium. To best accomplish this task, we circumvented the complexities of an actual porous catalyst by using a well defined porous matrix with uniform capillaric pores, i.e., a track-etched membrane. In this way, useful information was obtained regarding the relationship of molecular size and configuration on the diffusion rate of coal derived macromolecules through a pore structure with known geometry. Similar studies were performed using a pellet formed of porous alumina, to provide a link between the idealized membranes and the actual complex pore structure of real catalyst extrudates. The fundamental information from our study will be useful toward the tailoring of catalysts to minimize diffusional influences and thereby increase coal conversion and selectivity for desirable products. (VC)

  15. 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.

  16. DNA-Mediated Transfer of an RNA Polymerase II Gene: Reversion of the Temperature-Sensitive Hamster Cell Cycle Mutant TsAF8 by Mammalian DNA

    PubMed Central

    Ingles, C. James; Shales, Michael

    1982-01-01

    Treatment of the TsAF8 temperature-sensitive (TS) mutant of Syrian hamster BHK-21 cells, with calcium phosphate precipitates of genomic TS+ DNAs from a variety of mammalian cell lines permitted the selection of TS+ colonies at 40°C. TS+ transformation events were distinguished from spontaneous TS+ reversions in experiments in which ?-amanitin-sensitive (AmaS) TS+ DNA was used to transform an AmaR derivative of TsAF8 cells and AmaR TS+ DNA was used to transform AmaS TsAF8 cells. In each case it was possible to demonstrate the unselected acquisition of the appropriate AmaS or AmaR phenotype with the selected TS+ allele. Each of these TS+ transformed cell lines when grown at 40°C contained an RNA polymerase II activity with a sensitivity to inhibition by ?-amanitin characteristic of the particular DNA used to transform the TS cells, whereas at 34°C the same cells contained a mixture of AmaR and AmaS polymerase II activities. Together, these data provide convincing evidence that the RNA polymerase II gene determining sensitivity to inhibition by ?-amanitin can be transferred to TsAF8 cells and that the TS defect in TsAF8 is a polymerase II mutation. PMID:14582161

  17. Gbp1p, a Protein with RNA Recognition Motifs, Binds Single-Stranded Telomeric DNA and Changes Its Binding Specificity upon Dimerization

    PubMed Central

    Johnston, Stephen D.; Lew, Jodi E.; Berman, Judith

    1999-01-01

    Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed. PMID:9858616

  18. Total DNA transcription in vitro: a procedure to detect highly repetitive and transcribable sequences with tRNA-like structures

    SciTech Connect

    Endoh, H.; Okada, N.

    1986-01-01

    Total DNAs from various animals were transcribed in vitro in a HeLa cell extract, and it was found that one to several discrete RNAs were transcribed by RNA polymerase III. With tortoise (Geoclemys reevessi) and newt (Cynops pyrrhogaster), distinct 6.5S and 8S RNAs were transcribed from these respective DNAs. Representative phage clones carrying the 6.5S and 8S RNA genes were isolated from genomic libraries of these animals, and the sequences of these genes were determined. The 5' parts of highly repetitive and transcribable sequences of tortoise and newt were found to have close resemblance to tRNA/sub 1//sup Lys/ (rabbit) gene (78% homology) and a tRNA/sup Glu/ (Drosophila) gene (74% homology, not counting the aminoacyl stem region), respectively. The homologies extended to secondary structures, homologous nucleotides being located on similar secondary structures. It is proposed that many, if not all, highly repetitive and transcribable sequences detected by total DNA transcription have specific tRNA genes as their progenitors.

  19. 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

  20. Sequence heteroplasmy of D-loop and rRNA coding regions in mitochondrial DNA from Holstein cows of independent maternal lineages.

    PubMed

    Wu, J; Smith, R K; Freeman, A E; Beitz, D C; McDaniel, B T; Lindberg, G L

    2000-10-01

    A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy. PMID:11129526

  1. Pre-organized Guide RNA in the Cas9 Complex Is Ready for the Selection of Target Double-Stranded DNA.

    PubMed

    Kamiya, Yukiko; Asanuma, Hiroyuki

    2015-11-01

    The 3?D structure of Cas9 bound with sgRNA was solved by X-ray crystal-structure analysis. The conformation change in SpyCas9 upon binding to sgRNA changes Cas9 to target-DNA-recognition mode in which seed segments in sgRNA are pre-ordered in an A-type conformation. PMID:26300258

  2. Unique nucleocytoplasmic dsDNA and +ssRNA viruses are associated with the dinoflagellate endosymbionts of corals.

    PubMed

    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

  3. Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

    PubMed Central

    Wilson, Megan J.; Lamont, Iain L.

    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 region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function. PMID:16484205

  4. Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex

    SciTech Connect

    Zain, S.B.; Salim, M.; Chou, W.G.; Sajdel-Sulkowska, E.M.; Majocha, R.E.; Marotta, C.A.

    1988-02-01

    To gain insight into factors associated with the excessive accumulation of ..beta..-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the AD-specific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, the authors cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A)/sup +/ RNA from AD cortices was used for the preparation of lambdagt11 recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the ..beta..-amyloid domain and corresponded to 75% of the coding region and approx. = 70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, they conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

  5. A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Modulates 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

  6. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2?-deoxycytidine in human leukemic KG1 cells

    PubMed Central

    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.

    2015-01-01

    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

  7. 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

  8. Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae.

    PubMed

    Hookey, J V; Birtles, R J; Saunders, N A

    1995-09-01

    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

  9. Trypanosoma brucei gambiense Spliced Leader RNA Is a More Specific Marker for Cure of Human African Trypanosomiasis Than T. b. gambiense DNA.

    PubMed

    Ilboudo, Hamidou; Camara, Oumou; Ravel, Sophie; Bucheton, Bruno; Lejon, Veerle; Camara, Mamadou; Kaboré, Jacques; Jamonneau, Vincent; Deborggraeve, Stijn

    2015-12-15

    To assess the efficacy of treatment for human African trypanosomiasis, accurate tests that can discriminate relapse from cure are needed. We report the first data that the spliced leader (SL) RNA is a more specific marker for cure of human African trypanosomiasis than parasite DNA. In blood samples obtained from 61 patients in whom human African trypanosomiasis was cured, SL RNA detection had specificities of 98.4%-100%, while DNA detection had a specificity of only 77%. Data from our proof-of-concept study show that SL RNA detection has high potential as a test of cure. PMID:26080371

  10. A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

    PubMed Central

    Tagalakis, Aristides D; Castellaro, Sara; Zhou, Haiyan; Bienemann, Alison; Munye, Mustafa M; McCarthy, David; White, Edward A; Hart, Stephen L

    2015-01-01

    Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the ?-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo. PMID:25878500

  11. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    PubMed Central

    Boers, Stefan A.; Hays, John P.; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  12. Ultrasensitive Detection of MicroRNA in Tumor Cells and Tissues via Continuous Assembly of DNA Probe.

    PubMed

    Liao, Yuhui; Fu, Yu; Wu, Yunxia; Huang, Ru; Zhou, Xiaoming; Xing, Da

    2015-11-01

    Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analyte's initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis. PMID:26461520

  13. Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels.

    PubMed

    Vincent-Hubert, Françoise; Châtel, Amélie; Gourlay-Francé, Catherine

    2014-05-15

    We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification. PMID:24681118

  14. Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme.

    PubMed

    Chan, Laura; Tram, Kha; Gysbers, Rachel; Gu, Jimmy; Li, Yingfu

    2015-12-01

    We have previously shown that through test-tube molecular evolution, an arbitrarily chosen noncatalytic DNA sequence can be evolved into a catalytic DNA (DNAzyme) with significant RNA-cleaving activity. In this study, we aim to address the question of whether the catalytic activity of such a DNAzyme can be further optimized using in vitro selection. Several cycles of selective enrichment starting with a partially randomized DNA library have resulted in the isolation of many sequence variations that show notably improved catalytic activity. Bioinformatic analysis and activity examination of several DNAzyme-substrate constructs have led to two interesting findings about sequence mutations and the secondary structure of this DNAzyme: (1) three crucial mutations have transformed the DNAzyme into 8-17, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8-17 variant to make structural fine-tuning in order to cleave an arbitrarily chosen RNA-containing substrate with a defined sequence. Our study not only showcases the combined power of directed molecular evolution and in vitro selection techniques in turning a noncatalytic nucleic acid sequence into an efficient enzyme, but it also raises the question of whether mother nature has used a similar approach to evolve natural enzymes. PMID:26530076

  15. New roles for DNA cytosine modification, eRNA, anchors, and superanchors in developing B cell progenitors.

    PubMed

    Benner, Christopher; Isoda, Takeshi; Murre, Cornelis

    2015-10-13

    B-cell fate is orchestrated by a series of well-characterized developmental regulators. Here, we found that the onset of B-cell development was accompanied by large-scale changes in DNA cytosine modifications associated with promoters, enhancers, and anchors. These changes were tightly linked to alterations in transcription factor occupancy and nascent RNA (eRNA) transcription. We found that the prepro-B to the pro-B-cell transition was associated with a global exchange of DNA cytosine modifications for polycomb-mediated repression at CpG islands. Hypomethylated regions were found exclusively in the active/permissive compartment of the nucleus and were predominantly associated with regulatory elements or anchors that orchestrate the folding patterns of the genome. We identified superanchors, characterized by clusters of hypomethylated CCCTC-binding factor (CTCF)-bound elements, which were predominantly located at boundaries that define topological associated domains. A particularly prominent hypomethylated superanchor was positioned down-stream of the Ig heavy chain (Igh) locus. Analysis of global formaldehyde-cross-linking studies indicated that the Igh locus superanchor interacts with the VH region repertoire across vast genomic distances. We propose that the Igh locus superanchor sequesters the VH and DHJH regions into a spatial confined geometric environment to promote rapid first-passage times. Collectively, these studies demonstrate how, in developing B cells, DNA cytosine modifications associated with regulatory and architectural elements affect patterns of gene expression, folding patterns of the genome, and antigen receptor assembly. PMID:26417104

  16. "Parallel" and "antiparallel tail-clamps" increase the efficiency of triplex formation with structured DNA and RNA targets.

    PubMed

    Nadal, Anna; Eritja, Ramon; Esteve, Teresa; Pla, Maria

    2005-06-01

    Sequence-specific triple-helix structures can be formed by parallel and antiparallel DNA clamps interacting with single-stranded DNA or RNA targets. Single-stranded nucleic acid molecules are known to adopt secondary structures that might interfere with intermolecular interactions. We demonstrate the correlation between a secondary structure involving the target--a stable stem predicted by in silico folding and experimentally confirmed by thermal stability and competition analyses--and an inhibitory effect on triplex formation. We overcame structural impediments by designing a new type of clamp: "tail-clamps". A combination of gel-shift, kinetic analysis, UV thermal melting and thermodynamic techniques was used to demonstrate that tail-clamps efficiently form triple helices with a structured target sequence. The performance of parallel and antiparallel tail-clamps was compared: antiparallel tail-clamps had higher binding efficiencies than parallel tail-clamps both with structured DNA and RNA targets. In addition, the reported triplex-stabilizing property of 8-aminopurine residues was confirmed for tail-clamps. Finally, we discuss the possible use of this improved triplex technology as a new tool for applications in molecular biology. PMID:15880676

  17. Activated levels of rRNA synthesis in fission yeast are driven by an intergenic rDNA region positioned over 2500 nucleotides upstream of the initiation site.

    PubMed Central

    Liu, Z; Zhao, A; Chen, L; Pape, L

    1997-01-01

    RNA polymerase I-catalyzed synthesis of the Schizosaccharomyces pombe approximately 37S pre-rRNAs was shown to be sensitive to regulatory sequences located several kilobases upstream of the initiation site for the rRNA gene. An in vitro transcription system for RNA polymerase I-catalyzed RNA synthesis was established that supports correct and activated transcription from templates bearing a full S. pombe rRNA gene promoter. A 780 bp region starting at -2560 significantly stimulates transcription of ac is-located rDNA promoter and competes with an rDNA promoter in trans, thus displaying some of the activities of rDNA transcriptional enhancers in vitro. Deletion of a 30 bp enhancer-homologous domain in this 780 bp far upstream region blocked its cis-stimulatory effect. The sequence of the S. pombe 3.5 kb intergenic spacer was determined and its organization differs from that of vertebrate, Drosophila, Acanthamoeba and plant intergenic rDNA spacers: it does not contain multiple, imperfect copies of the rRNA gene promoter nor repetitive elements of 140 bp, as are found in vertebrate rDNA enhancers. PMID:9016610

  18. Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.

    PubMed

    Turchinovich, Andrey; Surowy, Harald; Serva, Andrius; Zapatka, Marc; Lichter, Peter; Burwinkel, Barbara

    2014-01-01

    Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called 'Capture and Amplification by Tailing and Switching' (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2-3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples. PMID:24922482

  19. 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

  20. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

    PubMed Central

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-01-01

    UVB irradiation induces harmful photochemical reactions, including formation of cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  1. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

    PubMed

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-08-25

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor ?(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires ?(S) and, together, RprA and ?(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer. PMID:26307765

  2. 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

  3. A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

    PubMed Central

    Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

    2015-01-01

    Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30?minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

  4. A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers.

    PubMed

    Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

    2015-01-01

    Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30?minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

  5. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D.

    PubMed

    Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S

    2012-12-21

    Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. PMID:23198659

  6. STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells.

    PubMed

    Liao, Xing-Hua; Zheng, Li; He, Hong-Peng; Zheng, De-Liang; Wei, Zhao-Qiang; Wang, Nan; Dong, Jian; Ma, Wen-Jian; Zhang, Tong-Cun

    2015-11-01

    Skin cancer is a major cause of morbidity and mortality worldwide. Mounting evidence shows that exposure of the skin to solar UV radiation results in inflammation, oxidative stress, DNA damage, dysregulation of cellular signaling pathways and immunosuppression thereby resulting in skin cancer. Signal transducer and activator of transcription 3 (STAT3) is well known to function as an anti-apoptotic factor, especially in numerous malignancies, but the relationship between STAT3 activation and DNA damage response in skin cancer is still not fully understood. We now report that STAT3 inhibited DNA damage induced by UV and STAT3 mediated upregulation of GADD45? and MDC-1 and the phosphorylation of H2AX in UV induced DNA damage. Notably, STAT3 can increase the expression of ATR in A431 cells. Luciferase assay shows that STAT3 activates the transcription of ATR promoter. More importantly, microRNA-383 suppressed ATR expression by targeting 3' (untranslated regions)UTR of ATR in A431 cells, and STAT3 down-regulates the transcription of miR-383 promoter. Thus, these results reveal the new insight that ATR is down-regulated by STAT3-regulated microRNA-383 in A431 cells. Moreover, overexpression of STAT3 enhanced expression of antiapoptosis genes BCL-1 and MCL-1, and depletion of STAT3 sensitized A431 cells to apoptotic cell death following UV. Collectively, these studies suggest that STAT3 may be a potential target for both the prevention and treatment of human skin cancer. PMID:26261078

  7. The interplay between DNA damage response and RNA processing: the unexpected role of splicing factors as gatekeepers of genome stability

    PubMed Central

    Naro, Chiara; Bielli, Pamela; Pagliarini, Vittoria; Sette, Claudio

    2015-01-01

    Genome integrity is constantly threatened by endogenous and exogenous factors. However, its preservation is ensured by a network of pathways that prevent and/or repair the lesion, which constitute the DNA damage response (DDR). Expression of the key proteins involved in the DDR is controlled by numerous post-transcriptional mechanisms, among which pre-mRNA splicing stands out. Intriguingly, several splicing factors (SFs) have been recently shown to play direct functions in DNA damage prevention and repair, which go beyond their expected splicing activity. At the same time, evidence is emerging that DNA repair proteins (DRPs) can actively sustain the DDR by acting as post-transcriptional regulator of gene expression, in addition to their well-known role in the mechanisms of signaling and repair of the lesion. Herein, we will review these non-canonical functions of both SFs and DRPs, which suggest the existence of a tight interplay between splicing regulation and canonical DNA safeguard mechanisms ensuring genome stability. PMID:25926848

  8. The interplay between DNA damage response and RNA processing: the unexpected role of splicing factors as gatekeepers of genome stability.

    PubMed

    Naro, Chiara; Bielli, Pamela; Pagliarini, Vittoria; Sette, Claudio

    2015-01-01

    Genome integrity is constantly threatened by endogenous and exogenous factors. However, its preservation is ensured by a network of pathways that prevent and/or repair the lesion, which constitute the DNA damage response (DDR). Expression of the key proteins involved in the DDR is controlled by numerous post-transcriptional mechanisms, among which pre-mRNA splicing stands out. Intriguingly, several splicing factors (SFs) have been recently shown to play direct functions in DNA damage prevention and repair, which go beyond their expected splicing activity. At the same time, evidence is emerging that DNA repair proteins (DRPs) can actively sustain the DDR by acting as post-transcriptional regulator of gene expression, in addition to their well-known role in the mechanisms of signaling and repair of the lesion. Herein, we will review these non-canonical functions of both SFs and DRPs, which suggest the existence of a tight interplay between splicing regulation and canonical DNA safeguard mechanisms ensuring genome stability. PMID:25926848

  9. 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

  10. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  11. DNA-mediated transfer of an RNA polymerase II gene: Reversion of the temperature-sensitive hamster cell cycle mutant TsAF8 by mammalian DNA

    SciTech Connect

    Ingles, C.J.; Shales, M.

    1982-06-01

    Treatment of the TsAF8 temperature-sensitive (TS) mutant of Syrian hamster BHK-21 cells, with calcium phosphate precipitates of genomic TS/sup +/ DNAs from a variety of mammalian cell lines permitted the selection of TS/sup +/ colonies at 40/sup 0/C. TS/sup +/ transformation events were distinguished from spontaneous TS/sup +/ reversions in experiments in which ..cap alpha..-amanitin-sensitive (Ama/sup s/) TS/sup +/ DNA was used to transform an AMA/sup R/ derivative of TsAF8 cells and Ama/sup R/ TS/sup +/ DNA was used to transform Ama/sup s/ TsAF8 cells. In each case it was possible to demonstrate the unselected acquisition of the appropriate Ama/sup s/ or Ama/sup R/ phenotype with the selected TS/sup +/ allele. Each of these TS/sup +/ transformed cell lines when grown at 40/sup 0/C contained an RNA polymerase II activity with a sensitivity to inhibition by ..cap alpha..-amanitin characteristic of the particular DNA used to transform the TS cells, whereas at 34/sup 0/C the same cells contained a mixture of Ama/sup R/ and Ama/sup s/ polymerase II activities. Together, these data provide convincing evidence that the RNA polymerase II gene determining sensitivity to inhibition by ..cap alpha..-amanitin can be transferred to TsAF8 cells and that the TS defect in TsAF8 is a polymerase II mutation.

  12. Edinburgh Research Explorer Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase

    E-print Network

    Millar, Andrew J.

    genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom Abstract During transcription

  13. 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 ...

  14. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing

    E-print Network

    Robinson, Gene E.

    on develop- ment and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here methylation is to regulate alternative splicing. epigenetics | gene regulation | gene silencing | insect One inherited, it is also environmentally responsive. DNA cytosine methylation is an epigenetic mechanism

  15. Coding DNA repeated throughout intergenic regions of the Arabidopsis thaliana genome: Evolutionary footprints of RNA silencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyknons are non-random sequence patterns significantly repeated throughout non-coding genomic DNA that also appear at least once among genes. They are interesting because they portend an unforeseen connection between coding and non-coding DNA. Pyknons have only been discovered in the human genome,...

  16. Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation.

    PubMed

    Santoro, Raffaella; Grummt, Ingrid

    2005-04-01

    Epigenetic control mechanisms silence about half of the rRNA genes in eukaryotes. Previous studies have demonstrated that recruitment of NoRC, a SNF2h-containing remodeling complex, silences rRNA gene transcription. NoRC mediates histone H4 deacetylation, histone H3-Lys9 dimethylation, and de novo DNA methylation, thus establishing heterochromatic features at the rRNA gene promoter. Here we show that inhibition of any of these activities alleviates NoRC-dependent silencing, indicating that these processes are intimately linked. We have studied the temporal order of epigenetic events at the rRNA gene promoter during gene silencing and demonstrate that recruitment of NoRC by TTF-I is a prerequisite for the deacetylation of histone H4 and the dimethylation of histone H3-Lys9. Inhibition of histone deacetylation prevents DNA methylation, while inhibition of DNA methylation does not affect histone modification. Importantly, ATP-dependent chromatin remodeling is required for methylation of a specific CpG dinucleotide within the upstream control element of the rRNA gene promoter, and this modification impairs preinitiation complex formation. The results of this study reveal a clear hierarchy of epigenetic events that control de novo DNA methylation and lead to silencing of RNA genes. PMID:15767661

  17. 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

  18. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  19. Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

    PubMed Central

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

  20. 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

  1. Nanomechanics of cartilage extracellular matrix macromolecules

    E-print Network

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

    2007-01-01

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

  2. Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

    NASA Technical Reports Server (NTRS)

    Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

    1993-01-01

    An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

  3. Whole-mount MeFISH: a novel technique for simultaneous visualization of specific DNA methylation and protein/RNA expression.

    PubMed

    Shiura, Hirosuke; Okamoto, Akimitsu; Sasaki, Hiroyuki; Abe, Kuniya

    2014-01-01

    To understand the spatiotemporal changes in cellular status that occur during embryonic development, it is desirable to detect simultaneously the expression of genes, proteins, and epigenetic modifications in individual embryonic cells. A technique termed methylation-specific fluorescence in situ hybridization (MeFISH) was developed recently that can visualize the methylation status of specific DNA sequences in cells fixed on a glass slide. Here, we adapted this glass slide-based MeFISH to the study of intact embryos, and established a method called whole-mount MeFISH. This method can be applied to any DNA sequences in theory and, as a proof-of-concept experiment, we examined the DNA methylation status of satellite repeats in developing mouse primordial germ cells, in which global DNA demethylation is known to take place, and obtained a result that was consistent with previous findings, thus validating the MeFISH method. We also succeeded in combining whole-mount MeFISH with immunostaining or RNA fluorescence in situ hybridization (RNA-FISH) techniques by adopting steps to retain signals of RNA-FISH or immunostaining after harsh denaturation step of MeFISH. The combined methods enabled the simultaneous visualization of DNA methylation and protein or RNA expression at single-cell resolution without destroying embryonic and nuclear structures. This whole-mount MeFISH technique should facilitate the study of the dynamics of DNA methylation status during embryonic development with unprecedented resolution. PMID:24755742

  4. 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

  5. In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets

    SciTech Connect

    Wang, Ying; Li, Zhaohui; Weber, Thomas J.; Hu, Dehong; Lin, Chiann Tso; Li, Jinghong; Lin, Yuehe

    2013-07-23

    Adenosine-5’-triphosphate (ATP) and guanosine-5’-triphosphate (GTP) are primary energy resources and function coordinately for numerous reactions such as microtubule assembly, insulin secretion and ion channel regulation. We have developed a novel DNA/RNA aptamer- graphene oxide nanosheet (GO-nS) sensing platform that can selectively and simultaneously detect ATP and GTP in live cells. A fluorescent tag is covalently attached to aptamers and fluorescence is quenched upon binding of aptamer to the GO-nS. Fluorescently tagged aptamers that selectively bind ATP or GTP were isolated from an aptamer library and were adsorbed onto GO-nS. Upon incubation with targets (ATP and/or GTP), the aptamers readily dissociated from GO-nS and the fluorescent signal was recovered. By covalently attaching fluorophores, both ATP and GTP sensing aptamers could be exploited to simultaneously visualize aptamer dissociation in live cells. In addition, the GO-nS appear to be biocompatible and protect the adsorbed DNA/RNA aptamers from enzymatic cleavage. Our results support the application of aptamer/GO-nS as a sensing platform for nucleotides in living cells and have implications for the development of additional sensor platforms for other bio-molecules that show selective interactions with aptamers and other biomarkers.

  6. 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

  7. The Effect of Sulfur Substitution on the Excited-State Dynamics of DNA and RNA Base Derivatives

    NASA Astrophysics Data System (ADS)

    Pollum, Marvin; Crespo-Hernández, Carlos E.

    2014-06-01

    Substitution of oxygen by a sulfur atom in the natural DNA and RNA bases gives rise to a family of derivatives commonly known as the thiobases. Upon excitation with UV radiation, the natural bases are able to quickly and efficiently dissipate the imparted energy as heat to their surroundings. Thiobases, on the other hand, relax into a long-lived triplet excited state in quantum yields that approach unity. This finding has both fundamental and biological relevance because the triplet state plays a foremost role in the photochemistry of the thiobases, this is especially important in the current medicinal applications of thiobase derivatives. Using femtosecond transient absorption spectroscopy, we are able uncover the ultrafast dynamics leading to the population of this reactive triplet state. In particular, I will present our results on how the site of sulfur substitution and the degree of substitution impact these dynamics and I will compare these experimental results to some recent computational work. Pinning down the excited-state dynamics of the thiobases is important to furthering the understanding of dynamics in natural DNA/RNA bases, as well as to the discovery of thiobase derivatives with desirable therapeutic properties. The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.

  8. G-Quadruplex Structures Formed by Expanded Hexanucleotide Repeat RNA and DNA from the Neurodegenerative Disease-Linked C9orf72 Gene Efficiently Sequester and Activate Heme

    PubMed Central

    Grigg, Jason C.; Shumayrikh, Nisreen; Sen, Dipankar

    2014-01-01

    The expansion of a (G4C2)n repeat within the human C9orf72 gene has been causally linked to a number of neurodegenerative diseases, most notably familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies have shown that the repeat expansion alters gene function in four ways, disrupting the gene's normal cellular roles and introducing toxic gain of function at the level of both DNA and RNA. (G4C2)n DNA, as well as the RNA transcribed from it, are found to fold into four-stranded G-quadruplex structures. It has been shown that the toxicity of the RNA G-quadruplexes, often localized in intracellular RNA foci, lies in their ability to sequester many important RNA binding proteins. Herein we propose that a distinct toxic property of such RNA and DNA G-quadruplexes from the C9orf72 gene may arise from their ability to bind and oxidatively activate cellular heme. We show that G-quadruplexes formed by both (G4C2)4 RNA and DNA not only complex tightly with heme but also enhance its intrinsic peroxidase and oxidase propensities. By contrast, the antisense (C4G2)4 RNA and DNA neither bind heme nor influence its oxidative activity. Curiously, the ability of C9orf72 DNA and transcripts to bind and activate heme mirror similar properties that have been reported for the A? peptide and its oligomers in Alzheimer's disease neurons. It is therefore conceivable that C9orf72 RNA G-quadruplex tangles play roles in sequestering intracellular heme and promoting oxidative damage in ALS and FTD analogous to those proposed for A? peptide and its tangles in Alzheimer's Disease. Given that neurodegenerative diseases in general are characterized by mitochondrial and respiratory malfunctions, the role of C9orf72 DNA and RNA in heme sequestration as well as its inappropriate activation in ALS and FTD neurons may warrant examination. PMID:25207541

  9. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    PubMed

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression. PMID:26611597

  10. Target-Catalyzed DNA Four-Way Junctions for CRET Imaging of MicroRNA, Concatenated Logic Operations, and Self-Assembly of DNA Nanohydrogels for Targeted Drug Delivery.

    PubMed

    Bi, Sai; Xiu, Bao; Ye, Jiayan; Dong, Ying

    2015-10-21

    Here we report a target-catalyzed DNA four-way junction (DNA-4WJ) on the basis of toehold-mediated DNA strand displacement reaction (TM-SDR), which is readily applied in enzyme-free amplified chemiluminescence resonance energy transfer (CRET) imaging of microRNA. In this system, the introduction of target microRNA-let-7a (miR-let-7a) activates a cascade of assembly steps with four DNA hairpins, followed by a disassembly step in which the target microRNA is displaced and released from DNA-4WJ to catalyze the self-assembly of additional branched junctions. As a result, G-quadruplex subunit sequences and fluorophore fluorescein amidite (FAM) are encoded in DNA-4WJ in a close proximity, stimulating a CRET process in the presence of hemin/K(+) to form horseradish peroxidase (HRP)-mimicking DNAzyme that catalyzes the generation of luminol/H2O2 chemiluminescence (CL), which further transfers to FAM. The background signal is easily reduced using magnetic graphene oxide (MGO) to remove unreacted species through magnetic separation, which makes a great contribution to improve the detection sensitivity and achieves a detection limit as low as 6.9 fM microRNA-let-7a (miR-let-7a). In addition, four-input concatenated logic circuits with an automatic reset function have been successfully constructed relying on the architecture of the proposed DNA-4WJ. More importantly, DNA nanohydrogels are self-assembled using DNA-4WJs as building units after centrifugation, which are driven by liquid crystallization and dense packaging of building units. Moreover, the DNA nanohydrogels are readily functionalized by incorporating with aptamers, bioimaging agents, and drug loading sites, which thus are served as efficient nanocarriers for targeted drug delivery and cancer therapy with high loading capacity and excellent biocompatibility. PMID:26420675

  11. Catabolism of tritiated thymidine by aquatic microbial communities and incorporation of tritium into RNA and protein

    SciTech Connect

    Brittain, A.M.; Karl, D.M. )

    1990-05-01

    The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. Nonspecific labeling was greatest in sediment samples, for which {>=}95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. We also evaluated the specificity of (2-{sup 3}H) adenine incorporation into adenylate residues in both RNA and DNA in parallel with the ({sup 3}H) thymidine experiments and compared the degree of nonspecific labeling by ({sup 3}H) adenine with that derived from ({sup 3}H)thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.

  12. Brickworx builds recurrent RNA and DNA structural motifs into medium- and low-resolution electron-density maps

    SciTech Connect

    Chojnowski, Grzegorz; Wale?, Tomasz; Pi?tkowski, Pawe?; Potrzebowski, Wojciech; Bujnicki, Janusz M.

    2015-03-01

    A computer program that builds crystal structure models of nucleic acid molecules is presented. 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.

  13. 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

  14. 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

  15. Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

    E-print Network

    Zukerberg, Lawrence R

    Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis ...

  16. Purification of the mRNA for chicken very low density lipoproteinII and molecular cloning of its full-length double-stranded cDNA.

    PubMed Central

    Wieringa, B; Roskam, W; Arnberg, A; van der Zwaag-Gerritsen, J; Ab, G; Gruber, M

    1979-01-01

    The mRNA coding for the small apo-Very Low Density Lipoprotein (apo-VLDLII) from chicken serum was highly enriched by oligo(dT) chromatography and preparative gel electrophoresis of estrogenised liver RNA. Double-stranded cDNA was synthesised by the subsequent actions of reverse transcriptase and DNA polymerase, and used for a preliminary characterisation of the structural gene. Molecular cloning of dC-tailed ds-cDNA into the Pst I site of plasmid pBR 322 yielded several recombinant clones. Five chimeric DNAs were selected and characterised by restriction enzyme mapping and electron microscopy of R-loops. At least two of them (pVLDLII 3.33 and pVLDLII 4.82) contain an almost full-length ds-transcript of VLDLII mRNA in which no more than 10-20 bases at the 5'- end are missing. Images PMID:230463

  17. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5?-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5?-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5?-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5?-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5?-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  18. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.

    PubMed

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5'-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5'-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5'-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5'-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5'-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  19. Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination

    PubMed Central

    Noto, Tomoko; Kataoka, Kensuke; Suhren, Jan H.; Hayashi, Azusa; Woolcock, Katrina J.; Gorovsky, Martin A.; Mochizuki, Kazufumi

    2015-01-01

    Summary Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production. PMID:26095658

  20. Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination.

    PubMed

    Noto, Tomoko; Kataoka, Kensuke; Suhren, Jan H; Hayashi, Azusa; Woolcock, Katrina J; Gorovsky, Martin A; Mochizuki, Kazufumi

    2015-07-16

    Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production. PMID:26095658

  1. DNA methylation mediated silencing of microRNA-145 is a potential prognostic marker in patients with lung adenocarcinoma

    PubMed Central

    Xia, Wenjie; Chen, Qiang; Wang, Jie; Mao, Qixing; Dong, Gaochao; Shi, Run; Zheng, YanYan; Xu, Lin; Jiang, Feng

    2015-01-01

    The molecular mechanism of down-regulated microRNA-145 (miR-145) expression in lung adenocarcinoma (LAC) remains largely unknown. We hypothesized that aberrant hyper-methylation of the CpG sites silenced the expression of miR-145 in LAC. In consideration of its pivotal role in LAC development and progression, we also evaluated the clinical utility of miR-145 as a prognostic marker. We assessed the DNA methylation status of the miR-145 promoter region in 20 pairs of LAC and the matched non-tumor specimens. We subsequently applied our own LAC tissue microarray containing 92 pairs of tumor and non-tumor tissues with long time follow-up records to evaluate whether miR-145 is a potential prognostic marker in LAC. The Sequenom EpiTYPER MassArray analysis showed that miR-145 was down-regulated in human LAC tissues accompanied by increased DNA methylation of its upstream region, which was further validated by the data from TCGA database. Significance was observed between miR-145 expression and clinic-pathologic parameters. Univariate and multivariate analysis revealed that miR-145 expression level was an independent risk factor for both OS and DFS in LAC patients. Taken together, DNA hyper-methylation in the miR-145 promoter region reduced its expression in LAC and miR-145 expression level might serve as a novel prognostic biomarker. PMID:26582602

  2. Calorimetry of Nanophases of Macromolecules

    NASA Astrophysics Data System (ADS)

    Wunderlich, Bernhard

    2007-06-01

    The thermodynamic description of polymeric systems is summarized based on 50 years of gathering experimental information with adiabatic, differential-scanning, and temperature-modulated calorimetry. This experience has led to a description of macro- to micro- to nano-phases with macromolecules able to traverse the phase boundaries and decouple at the surfaces, resulting in different thermodynamic properties for the separated parts of the molecule. A typical thermodynamic characterization of semicystalline polymers is that of a globally metastable system with locally reversible processes. Unique phenomena in polymers include the ability of semicrystalline polymers to undergo cold crystallization and molecular nucleation, possess thermally generated point defects and rigid-amorphous fractions, and have amorphous to mesophasic to crystalline macroconformations with glass, ordering, and disordering transitions in all three structures. To describe such multifaceted systems, special combinations of equilibrium, and irreversible thermodynamics as well as statistical and quantum mechanics are necessary. Only then is it possible to handle violations of phase rules, changes of properties when approaching nanophase dimensions, local reversibility, and enthalpy relaxation. The enthalpy relaxation in polymers originates in the cooperativeness of conformational motion and the interferences of processes of different time scales. The experiments to identify the effects of the different molecular motions from typical vibrational time scales of picoseconds to cooperative, large-amplitude rearrangements of up to megaseconds span heating rates of thousands of K·s-1 with superfast chip calorimeters to many hours for slow, quasi-isothermal analysis by temperature-modulated differential scanning calorimetry (TMDSC). Selected examples of this far-reaching thermal characterization will be presented.

  3. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

    PubMed Central

    Carroll, Paul; Donatello, Simona; Connolly, Elizabeth; Griffin, Mairead; Dunne, Barbara; Burke, Louise; Flavin, Richard; Rizkalla, Hala; Ryan, Ciara; Hayes, Brian; D'Adhemar, Charles; Banville, Niamh; Faheem, Nazia; Muldoon, Cian; Gaffney, Eoin F.

    2011-01-01

    The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep® (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy® and QIAamp® (both Qiagen), and (4) to examine the effectiveness of Allprotect® (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software®). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A260/280 values using QIAamp were 33.2?ng/?L and 1.86, respectively, and using AllPrep were 23.2?ng/?L and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4?ng/?L and 8.16, respectively, and with AllPrep were 74.8?ng/?L and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6?h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates. PMID:23386926

  4. Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples.

    PubMed

    Mee, Blanaid C; Carroll, Paul; Donatello, Simona; Connolly, Elizabeth; Griffin, Mairead; Dunne, Barbara; Burke, Louise; Flavin, Richard; Rizkalla, Hala; Ryan, Ciara; Hayes, Brian; D'Adhemar, Charles; Banville, Niamh; Faheem, Nazia; Muldoon, Cian; Gaffney, Eoin F

    2011-12-01

    The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2?ng/?L and 1.86, respectively, and using AllPrep were 23.2?ng/?L and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4?ng/?L and 8.16, respectively, and with AllPrep were 74.8?ng/?L and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6?h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates. PMID:23386926

  5. U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA.

    PubMed

    Wong, Richard G; Kazane, Katelynn; Maslov, Dmitri A; Rogers, Kestrel; Aphasizhev, Ruslan; Simpson, Larry

    2015-11-01

    We studied the intramitochondrial localization of several multiprotein complexes involved in U-insertion/deletion RNA editing in trypanosome mitochondria. The editing complexes are located in one or two antipodal nodes adjacent to the kinetoplast DNA (kDNA) disk, which are distinct from but associated with the minicircle catenation nodes. In some cases the proteins are in a bilateral sheet configuration. We also found that mitoribosomes have a nodal configuration. This type of organization is consistent with evidence for protein and RNA interactions of multiple editing complexes to form an ~40S editosome and also an interaction of editosomes with mitochondrial ribosomes. PMID:26462764

  6. BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes

    PubMed Central

    Alekseyenko, Artyom A.; McElroy, Kyle A.; Kang, Hyuckjoon; Zee, Barry M.; Kharchenko, Peter V.; Kuroda, Mitzi I.

    2015-01-01

    In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components and their mutual interactions. Here we present a chromatin cross-linking approach (BioTAP-XL) which utilizes mass-spectrometry to identify protein complex components, together with high-throughput sequencing to identify RNA components and DNA binding sites. We describe full protocols for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of our approach in all cases is the generation of control data from input chromatin samples. PMID:25559106

  7. Binding to DNA of the RNA-polymerase II C-terminal domain allows discrimination between Cdk7 and Cdk9 phosphorylation

    PubMed Central

    Lolli, Graziano

    2009-01-01

    The C-terminal domain (CTD) of RNA polymerase II regulates transcription through spatially and temporally coordinated events. Previous work had established that the CTD binds DNA but the significance of this interaction has not been determined. The present work shows that the CTD binds DNA in its unphosphorylated form, the form in which it is present in the pre-initiation complex. The CTD/DNA complex is recognized by and is phosphorylated by Cdk7 but not by Cdk9. Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex. The model has been tested by mutagenesis experiments. CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation. The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation. PMID:19136461

  8. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. While investigating virus-invertebrate host interactions we found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), were unable to infect certain Lepido...

  9. Terminal strand-switching of E. coli RNA polymerase transcribing a truncated DNA fragment.

    PubMed

    Oostra, B A; Arnberg, A C; Ab, G; Gruber, M

    1981-10-27

    When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch. PMID:6269631

  10. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    PubMed Central

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1?, p65/NF-?B), the angiogenic factor bFGF, inflammatory factors (IL-1?, IL-6, TNF?, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNF? from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-?B, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation. PMID:26330750

  11. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA

    PubMed Central

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-01-01

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. PMID:25999340

  12. Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

    PubMed Central

    Sherman, M P; Ehrlich, G D; Ferrer, J F; Sninsky, J J; Zandomeni, R; Dock, N L; Poiesz, B

    1992-01-01

    Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine. Images PMID:1370847

  13. Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models.

    PubMed

    Grabmüller, Melanie; Schyma, Christian; Euteneuer, Jan; Madea, Burkhard; Courts, Cornelius

    2015-09-01

    When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics. PMID:26210238

  14. Conformational studies of chiral D-Lys-PNA and achiral PNA system in binding with DNA or RNA through a molecular dynamics approach.

    PubMed

    Autiero, Ida; Saviano, Michele; Langella, Emma

    2015-02-16

    The growing interest in peptide nucleic acid (PNA) oligomers has led to the development of a very wide variety of PNA derivatives. Among others, the introduction of charged chiral groups on a PNA oligomer has proven effective in improving DNA binding ability, complexation direction and cellular uptake. In particular, the introduction of three adjacent chiral monomers based on D-Lys in the middle of the PNA sequence (D-Lys-PNA) has produced noteworthy results in modulating the directionality of the binding with the DNA complementary strand and in mismatch detection. Here, through a molecular dynamics approach, a comparative study has been carried out to investigate the structural properties that drive the interaction of the chiral D-Lys-PNA and the corresponding achiral PNA system with DNA as well as RNA complementary strands, starting from the crystal structure of D-Lys-PNA in complex with DNA. The results obtained complement experimental data and indicate that the binding with the RNA molecule, compared to DNA, is differently affected by the addition of three D-Lys groups on the PNA backbone, suggesting that this modification could be taken into account for the development of new PNA-based molecules able to discriminate between DNA and RNA. PMID:25112690

  15. Increased rDNA synthesis in germinated conidia of Neurospora crassa is caused by RNA primer molecules found in its culture medium

    SciTech Connect

    Dutta, S.K.; Beljanski, M.

    1984-01-01

    Purine rich small primer RNA molecules (10-15 nucleotides) were isolated from growth medium of germinated (3 hr sprout) conidia of N. crassa. These RNA-primer molecules strongly stimulated in vitro DNA synthesis in N. crassa 74A wild type, as well as in DNAs from mice spleen and lung, and quail testis. These increases of in vitro DNA synthesis was dependent on the concentration of these RNA primer molecules. In contrast, such molecules were not found in 1 or 10 hour sprouts, nor in the culture medium of mycelia (24 hr). These RNA-primer molecules could be hydrolyzed by T1 RNAse but not by pancreatic RNase. Dutta et al. reported increased (250) copies of rRNA genes in germinated conidia (3 hr sprouts) compared to 100 copies of rRNA genes in mycelial cells grown for 24 hours. These observations suggest excessive transcription of rDNAs in the germinated conidial cells which undergo cleavages by nucleates after 3-4 hours of cell growth. Some degradation products were excreted into the culture medium and acted as RNA-primers.

  16. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    PubMed Central

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below ?10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  17. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses.

    PubMed

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  18. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DOE PAGESBeta

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy numbermore »of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.« less

  19. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    SciTech Connect

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  20. X-ray Crystallographic Observation of 'In-line' and 'Adjacent' Conformations in a Bulged Self-Cleaving RNA/DNA Hybrid

    SciTech Connect

    Tereshko, V.; Wallace, S.T.; Usman, N.; Wincott, F.; Egli, M.

    2010-03-08

    The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Husken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line ) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.

  1. Germ Line Basis for Antibody Diversity: Immunoglobulin VH- and CH-gene Frequencies Measured by DNA·RNA Hybridization

    PubMed Central

    Premkumar, E.; Shoyab, M.; Williamson, A. R.

    1974-01-01

    The reiteration frequency for mouse immunoglobulin VH-genes and CH-genes has been directly estimated by hybridization of purified MOPC 315 ?-chain mRNA with a vast excess of mouse DNA. A biphasic Cot curve resulted. The low Cot transition (Cot1/2 about 1.5) was interpreted as hybridization to VH-genes and the high Cot transition (Cot1/2 about 103) as hybridization to CH-genes. These values correspond to about 5000 VH-genes and less than 8 CH-genes. This germ-line content of VH-genes is sufficient to account for antibody diversity given a comparable set of VL-genes. Minimal-gene models are invalidated and there is no need to invoke somatic generators of diversity. PMID:4521059

  2. Unfolding and Melting of DNA (RNA) Hairpins: The Concept of Structure-Specific 2D Dynamic Landscapes

    PubMed Central

    Lin, Milo M.; Meinhold, Lars; Shorokhov, Dmitry; Zewail, Ahmed H.

    2008-01-01

    Summary A 2D free energy landscape model is presented to describe the (un)folding transition of DNA/RNA hairpins, together with molecular dynamics simulations and experimental findings. The dependence of the (un)folding transition on the stem sequence and the loop length is shown in the enthalpic and entropic contributions to the free energy. Intermediate structures are well defined by the two coordinates of the landscape during (un)zipping. Both the free energy landscape model and the extensive molecular dynamics simulations totaling over 10 ?s predict the existence of temperature-dependent kinetic intermediate states during hairpin (un)zipping and provide the theoretical description of recent ultrafast temperature-jump studies which indicate that hairpin (un)zipping is, in general, not a two-state process. The model allows for lucid prediction of the collapsed state(s) in simple 2D space and we term it the kinetic intermediate structure (KIS) model. PMID:18633543

  3. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    PubMed

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging. PMID:26548353

  4. Rationally Designed Small Molecules That Target Both the DNA and RNA Causing Myotonic Dystrophy Type 1.

    PubMed

    Nguyen, Lien; Luu, Long M; Peng, Shaohong; Serrano, Julio F; Chan, H Y Edwin; Zimmerman, Steven C

    2015-11-11

    Single-agent, single-target therapeutic approaches are often limited by a complex disease pathobiology. We report rationally designed, multi-target agents for myotonic dystrophy type 1 (DM1). DM1 originates in an abnormal expansion of CTG repeats (CTG(exp)) in the DMPK gene. The resultant expanded CUG transcript (CUG(exp)) identified as a toxic agent sequesters important proteins, such as muscleblind-like proteins (MBNL), undergoes repeat-associated non-ATG (RAN) translation, and potentially causes microRNA dysregulation. We report rationally designed small molecules that target the DM1 pathobiology in vitro in three distinct ways by acting simultaneously as transcription inhibitors, by inhibiting aberrant protein binding to the toxic RNA, and by acting as RNase mimics to degrade the toxic RNA. In vitro, the agents are shown to (1) bind CTG(exp) and inhibit formation of the CUG(exp) transcript, (2) bind CUG(exp) and inhibit sequestration of MBNL1, and (3) cleave CUG(exp) in an RNase-like manner. The most potent compounds are capable of reducing the levels of CUG(exp) in DM1 model cells, and one reverses two separate CUG(exp)-induced phenotypes in a DM1 Drosophila model. PMID:26473464

  5. Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein †

    PubMed Central

    Brittain, Andrew M.; Karl, David M.

    1990-01-01

    The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ?95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-3H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [3H]thymidine experiments and compared the degree of nonspecific labeling by [3H]adenine with that derived from [3H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples. PMID:16348180

  6. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    PubMed

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  7. Pse-in-One: a web server for generating various modes of pseudo components of DNA, RNA, and protein sequences.

    PubMed

    Liu, Bin; Liu, Fule; Wang, Xiaolong; Chen, Junjie; Fang, Longyun; Chou, Kuo-Chen

    2015-07-01

    With the avalanche of biological sequences generated in the post-genomic age, one of the most challenging problems in computational biology is how to effectively formulate the sequence of a biological sample (such as DNA, RNA or protein) with a discrete model or a vector that can effectively reflect its sequence pattern information or capture its key features concerned. Although several web servers and stand-alone tools were developed to address this problem, all these tools, however, can only handle one type of samples. Furthermore, the number of their built-in properties is limited, and hence it is often difficult for users to formulate the biological sequences according to their desired features or properties. In this article, with a much larger number of built-in properties, we are to propose a much more flexible web server called Pse-in-One (http://bioinformatics.hitsz.edu.cn/Pse-in-One/), which can, through its 28 different modes, generate nearly all the possible feature vectors for DNA, RNA and protein sequences. Particularly, it can also generate those feature vectors with the properties defined by users themselves. These feature vectors can be easily combined with machine-learning algorithms to develop computational predictors and analysis methods for various tasks in bioinformatics and system biology. It is anticipated that the Pse-in-One web server will become a very useful tool in computational proteomics, genomics, as well as biological sequence analysis. Moreover, to maximize users' convenience, its stand-alone version can also be downloaded from http://bioinformatics.hitsz.edu.cn/Pse-in-One/download/, and directly run on Windows, Linux, Unix and Mac OS. PMID:25958395

  8. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

    SciTech Connect

    Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

    2010-11-01

    Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m{sup 3}) and high (above 50 mg/m{sup 3}) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 {+-} 1.00 SSB/10{sup 9} Da), followed by high exposure group (0.72 {+-} 0.81 SSB/10{sup 9} Da) and controls (0.65 {+-} 0.82 SSB/10{sup 9} Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

  9. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

    PubMed

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

    2015-11-17

    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 ?m × 14 ?m and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools. PMID:26494264

  10. Enzyme-Free Amplification by Nano Sticky Balls for Visual Detection of ssDNA/RNA Oligonucleotides.

    PubMed

    Chen, Shan; Chu, Lok Ting; Yeung, Pak Piu; Zhao, Zichen; Bao, Yuanye; Chan, Miu Shan; Lo, Pik Kwan; Chen, Ting-Hsuan

    2015-10-21

    Visual detection of nucleic acids provides simple and rapid screening for infectious diseases or environmental pathogens. However, sensitivity is the current bottleneck, which may require enzymatic amplification for targets in low abundance and make them incompatible with detection at resource-limited sites. Here we report an enzyme-free amplification that provides a sensitive visual detection of ssDNA/RNA oligonucleotides on the basis of nano "sticky balls". When target oligonucleotides are present, magnetic microparticles (MMPs) and gold nanoparticles (AuNPs) were linked together, allowing the collection of AuNPs after magnetic attraction. Subsequently, the collected AuNPs, which carry many oligonucleotides, were used as the sticky balls to link a second pair of MMPs and polymer microparticles (PMPs). Thus, because the magnetic field can attract the MMPs as well as the linked PMPs to the sidewall, the reduction of suspended PMPs yields a change of light transmission visible by the naked eye. Our results demonstrate that the limit of detection is 10 amol for ssDNAs (228 fM in 45 ?L) and 75 amol for ssRNAs (1.67 pM in 45 ?L). This method is also compatible with the serum environment and detection of a microRNA, miR-155, derived from human breast cancer cells. With significantly improved sensitivity for visual detection, it provides great potential for point-of-care applications at resource-limited sites. PMID:26430877

  11. A General and Rapid Cell-Free Approach for the Interrogation of Protein-Protein, Protein-DNA, and Protein-RNA

    E-print Network

    Ghosh, Indraneel

    A General and Rapid Cell-Free Approach for the Interrogation of Protein-Protein, Protein-DNA, and Protein-RNA Interactions and their Antagonists Utilizing Split-Protein Reporters Jason R. Porter, Cliff I, Arizona 85721 Received December 28, 2007; E-mail: ghosh@email.arizona.edu Abstract: Split-protein

  12. Supplemental protocol for "Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins" (McNaughton, Cronican, Thompson,

    E-print Network

    Liu, David R.

    tissue culture plate. 1. HeLa cells were grown to ~ 80% confluency in a 12-well plate. DMEM / 10% FBS µM PBA/PBS. Much higher cytotoxicity (~80%) was observed when HeLa cells were incubated in 50 µM PBASupplemental protocol for "Mammalian cell penetration, siRNA transfection, and DNA transfection

  13. ASSESSING HABITAT QUALITY OF MOUNT HOPE BAY AND NARRAGANSETT BAY USING GROWTH, RNA:DNA, AND FEDDING HABITS OF CAGED JUVENILE WINTER FLOUNDER

    EPA Science Inventory

    Somatic growth rates, RNA:DNA, and feeding habits of juvenile Pseudopleuronectes americanus (Winter Flounder) were used to asses small-scale spatio-temporal variations in the habitat quality of Mount Hope Bay and Narragan-sett Bay, RI. Three successive caging experiments (14–16 d...

  14. High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips.

    PubMed

    Lu, Wenjing; Wang, Jidong; Wu, Qiong; Sun, Jiashu; Chen, Yiping; Zhang, Lu; Zheng, Chunsheng; Gao, Wenna; Liu, Yi; Jiang, Xingyu

    2016-01-15

    We develop a micro-pipette tip-based nucleic acid test (MTNT) for high-throughput sample-to-answer detection of both DNA and RNA from crude samples including cells, bacteria, and solid plants, without the need of sample pretreatment and complex operation. MTNT consists of micro-pipette tips and embedded solid phase nucleic acid extraction membranes, and fully integrates the functions of nucleic acid extraction from crude samples, loop-mediated isothermal amplification (LAMP) of nucleic acids, and visual readout of assays. The total assaying time for DNA or RNA from a variety of crude samples ranges from 90 to 160 min. The limit of detection (LOD) of MTNT is 2 copies of plasmids containing the target nucleic acid fragments of Ebola virus, and 8 CFU of Escherichia coli carrying Ebola virus-derived plasmids. MTNT can also detect CK-19 mRNA from as few as 2 cancer cells without complicated procedures such as RNA extraction and purification. We further demonstrate MTNT in a high-throughput format using an eight-channel pipette and a homemade mini-heater, with a maximum throughput of 40 samples. Compared with other point-of-care (POC) nucleic acid tests (NAT), MTNT could assay both DNA and RNA directly from liquid (cells/bacteria/blood) or solid (plant) samples in a straightforward, sensitive, high-throughput, and containment-free manner, suggesting a considerable promise for low-cost and POC NAT in remote areas. PMID:26283588

  15. Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

    PubMed

    Qi, Y; Zhou, X; Li, D

    2000-01-01

    The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully. PMID:10949946

  16. Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

    PubMed Central

    Czajka, J; Bsat, N; Piani, M; Russ, W; Sultana, K; Wiedmann, M; Whitaker, R; Batt, C A

    1993-01-01

    Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated. Images PMID:8439157

  17. Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations.

    PubMed Central

    Pluen, A; Netti, P A; Jain, R K; Berk, D A

    1999-01-01

    The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers. PMID:10388779

  18. Computational Analysis of LncRNA from cDNA Sequences.

    PubMed

    Boerner, Susan; McGinnis, Karen M

    2016-01-01

    Based on recent findings, long noncoding (lnc) RNAs represent a potential class of functional molecules within the cell. In this chapter we describe a computational scheme to identify and classify lncRNAs within maize from full-length cDNA sequences to designate subsets of lncRNAs for which biogenesis and regulatory mechanisms may be verified at the bench. We make use of the Coding Potential Calculator and specific Python scripts in our approach. PMID:26721497

  19. Trial by fire: are the crystals macromolecules?

    PubMed

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

    2010-05-01

    Protein crystallization screens frequently yield salt crystals as well as protein crystals. A simple method for determining whether a crystal is composed of salt or macromolecules is suggested. A drop containing one or more crystals is transferred to a glass cover slip and the cover slip is then passed through the flame of a Bunsen burner. Macromolecule crystals are destroyed by this treatment, while salt crystals generally remain. The test can be performed after other commonly used tests such as crushing and staining. PMID:20445273

  20. General Differential Contact Identities for Macromolecules

    NASA Astrophysics Data System (ADS)

    Landy, Jonathan; Pincus, P. A.; Jho, YongSeok

    2015-10-01

    We discuss general Maxwell identities relating a macromolecule's charge, the forces acting at its surface, and the osmotic pressure of the solution in which it sits. The identities are closely related to the contact value relations that hold for certain special geometries, but are more general. In particular, the Maxwell identities can be applied to any macromolecule geometry, and they hold both within and outside of mean-field theory. Examples illustrate that combining the identities with approximate treatments of screening can often return simple, accurate osmotic pressure estimates.

  1. Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi.

    PubMed

    Paterson, R R M; Lima, N

    2009-04-01

    Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus, as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers. PMID:19291250

  2. Downregulation of microRNA-132 by DNA hypermethylation is associated with cell invasion in colorectal cancer

    PubMed Central

    Qin, Jun; Ke, Jing; Xu, Junfei; Wang, Feiran; Zhou, Youlang; Jiang, Yasu; Wang, Zhiwei

    2015-01-01

    microRNAs (miRNAs) are small, noncoding RNAs that are involved in many biological processes, and aberrant regulation of miRNAs is always associated with cancer progression and development. Abnormal expression of miRNA-132 (miR-132) has been found in some types of cancer, but the effects and potential mechanisms of miR-132 in colorectal cancer (CRC) have not been explored to date. In this study, quantitative real-time polymerase chain reaction was used to investigate the level of miR-132 in CRC tissues and their paired adjacent normal tissues. Bioinformatics analysis indicated that the mechanism underlying the tumor suppressor role of miR-132 in CRC cells may play a role in tumor suppression by targeting paxillin. Furthermore, methylation-specific polymerase chain reaction was performed to evaluate the methylation status of the miR-132 regulatory region. A DNA methyltransferase inhibitor, 5-aza-2?-deoxycytidine, was used to activate the expression of miR-132 in CRC cells in vitro. Downregulation of miR-132 may occur as a result of hypermethylation and implies a poor prognosis in CRC; therefore, triggering miR-132 reexpression by using DNA methyltransferase inhibitors may be a potential molecular therapeutic target for CRC. PMID:26675712

  3. Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

    PubMed

    Felleisen, R S; Lambelet, N; Bachmann, P; Nicolet, J; Müller, N; Gottstein, B

    1998-02-01

    Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples. PMID:9466768

  4. RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus

    PubMed Central

    Huo, Longfei; Wang, Ying-Nai; Xia, Weiya; Hsu, Sheng-Chieh; Lai, Chien-Chen; Li, Long-Yuan; Chang, Wei-Chao; Wang, Yan; Hsu, Ming-Chuan; Yu, Yung-Luen; Huang, Tzu-Hsuan; Ding, Qingqing; Chen, Chung-Hsuan; Tsai, Chang-Hai; Hung, Mien-Chie

    2010-01-01

    EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR–RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus. PMID:20802156

  5. Ionization and fragmentation of DNA, RNA bases induced by proton impact

    NASA Astrophysics Data System (ADS)

    LePadellec, A.; Moretto-Capelle, P.; Richard-Viard, M.; Champeaux, J. P.; Cafarelli, P.

    2008-02-01

    We present recent results obtained in the Toulouse's group that deal with proton to base and nucleoside interactions. We stress the weakness of the sugar part in the nucleoside, i.e. the uridine molecule under scrutiny. Since some parts of the fragmentation spectrum correspond to the fragmentation of a 'pure' uracil molecule, i.e. the RNA base, an 'additivity rule' seems to prevail for the nucleoside, something that still has to be confirmed. Moreover, some results that deal with the secondary electronic emission from uracil are also displayed.

  6. Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

    PubMed

    Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

    2015-10-01

    The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. PMID:26078123

  7. Mycobacterium tuberculosis DNA Increases Vitamin D Receptor mRNA Expression and the Production of Nitric Oxide and Cathelicidin in Human Monocytes

    PubMed Central

    SISWANTO, Siswanto; ZUHRIYAH, Lilik; HANDONO, Kusworini; FITRI, Loeki Enggar; PRAWIRO, Sumarno Reto

    2015-01-01

    Background: The innate immune response to tuberculosis infection may involve the increased production of nitric oxide and cathelicidin due to the up-regulated expression of the vitamin D receptor (VDR), though this proposed mechanism remains controversial. The aim of this study was to determine how the exposure of human monocytes to Mycobacterium tuberculosis (M. tuberculosis) DNA affects the production of nitric oxide and cathelicidin, as well as the expression of VDR. Methods: This study was performed using monocytes obtained from healthy donors. After 24 h incubation, monocytes were stimulated with M. tuberculosis DNA for 18 h to determine the expression of VDR mRNA and the production of nitric oxide and cathelicidin versus non-stimulated cells (the control group). Results: The expression of VDR mRNA was higher in the monocytes exposed to M. tuberculosis DNA compared to the control group (P = 0.020). Monocytes exposed to M. tuberculosis DNA also showed significantly increased production of nitric oxide and cathelicidin compared to the control group (P = 0.0001; P = 0.028). Conclusion: The stimulation of human monocytes with M. tuberculosis DNA increases the expression of the VDR mRNA and the production of nitric oxide and cathelicidin. PMID:26715892

  8. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

    PubMed Central

    Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R.; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M.

    2013-01-01

    Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility (“retrohoming”) by a process that requires reverse transcription of a highly structured, 2–2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3? ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications. PMID:23697550

  9. Co-expression of miRNA targeting the expression of PERK, but not PKR, enhances cellular immunity from an HIV-1 Env DNA vaccine.

    PubMed

    Wheatley, Adam K; Kramski, Marit; Alexander, Marina R; Toe, Jesse G; Center, Rob J; Purcell, Damian F J

    2011-01-01

    Small non-coding micro-RNAs (miRNA) are important post-transcriptional regulators of mammalian gene expression that can be used to direct the knockdown of expression from targeted genes. We examined whether DNA vaccine vectors co-expressing miRNA with HIV-1 envelope (Env) antigens could influence the magnitude or quality of the immune responses to Env in mice. Human miR-155 and flanking regions from the non-protein encoding gene mirhg155 were introduced into an artificial intron within an expression vector for HIV-1 Env gp140. Using the miR-155-expressing intron as a scaffold, we developed novel vectors for miRNA-mediated targeting of the cellular antiviral proteins PKR and PERK, which significantly down-modulated target gene expression and led to increased Env expression in vitro. Finally, vaccinating BALB/c mice with a DNA vaccine vector delivering miRNA targeting PERK, but not PKR, was able to augment the generation of Env-specific T-cell immunity. This study provides proof-of-concept evidence that miRNA effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Further testing of vaccine-encoded miRNA will determine if such strategies can enhance protective efficacy from vaccines against HIV-1 for eventual human use. PMID:21464971

  10. Dependence of RNA:DNA ratios and Fulton’s K condition indices on environmental characteristics of plaice and dab nursery grounds

    NASA Astrophysics Data System (ADS)

    De Raedemaecker, F.; Brophy, D.; O'Connor, I.; O'Neill, B.

    2012-02-01

    This field study showed a lack of a correlation between a morphometric (Fulton's K) and biochemical (RNA:DNA ratio) condition index in juvenile plaice ( Pleuronectes platessa) and dab ( Limanda limanda) studied to assess habitat quality in four sandy beach nursery grounds in Galway Bay, Ireland. Based on monthly surveys from June to September in 2008 and 2009, fish growth, indicated by RNA:DNA ratios and Fulton's K, displayed considerable spatio-temporal variability. Site-related patterns in Fulton's K for plaice and dab were consistent between years whereas RNA:DNA ratios displayed annual and interspecific variability among nursery habitats. This indicates a higher sensitivity of RNA:DNA ratios to short-term environmental fluctuations which is not apparent in Fulton's K measurements of juvenile flatfish. Generalized Additive Modelling (GAM) revealed non-linear relationships between the condition indices and (biotic and abiotic) habitat characteristics as well as diet features, derived from gut content analyses. Density of predators, sediment grain size and salinity were the most important predictors of both condition indices. Temperature also affected condition indices in dab whereas plaice condition indices varied with depth. Diet features did not contribute to the explained variability in the models predicting RNA:DNA ratios whereas certain prey groups significantly improved the explained variability in the models predicting Fulton's K of plaice and dab. The value of both indices for assessing fish condition and habitat quality in field studies is discussed. These findings aid understanding of the biological and physical mechanisms promoting fast growth and high survival which will help to identify high quality nursery areas for juvenile plaice and dab.

  11. A High-Throughput Macromolecule Characterization System

    E-print Network

    Kim, Jae Hyun

    2013-08-31

    -throughput systems, in terms of both hardware and software, for biophysical characterization of macromolecules. For this purpose, a web-based software framework called MiddaughSuite was developed in this work. The software was designed to easily handle data from...

  12. The nucleotide sequences of the regions flanking the genes coding for 23S, 16S and 4.5S ribosomal RNA on chloroplast DNA from Spirodela oligorhiza.

    PubMed Central

    Keus, R J; Dekker, A F; van Roon, M A; Groot, G S

    1983-01-01

    The nucleotide sequences of the flanking regions of the genes coding for Spirodela oligorhiza chloroplast ribosomal RNA's have been determined. We have compared these sequences to the corresponding ones in chloroplast DNA of other plants and of E. coli and find a striking sequential or structural homology. The region 5'-proximal to the gene coding for 16S rRNA contains a gene coding for tRNAval, which is transcribed from the same strand. In this area three prokaryotic promoter motifs are found: one located in front of the tRNAval gene and two in the intergenic space between this gene and the 16S rRNA gene. The middle one is used for the start of the transcription of the large ribosomal RNA precursor. Images PMID:6312425

  13. Macromolecules Inquiry: Transformation of a Standard Biochemistry Lab

    ERIC Educational Resources Information Center

    Unsworth, Elizabeth

    2014-01-01

    Identification of macromolecules in food is a standard introductory high school biology lab. The intent of this article is to describe the conversion of this standard cookbook lab into an inquiry investigation. Instead of verifying the macromolecules found in food, students use their knowledge of the macromolecules in food to determine the…

  14. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. PMID:26245685

  15. tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum.

    PubMed Central

    Preiser, P; Williamson, D H; Wilson, R J

    1995-01-01

    Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle. Images PMID:7501453

  16. ABCB6 mRNA and DNA methylation levels serve as useful biomarkers for prediction of early intrahepatic recurrence of hepatitis C virus-related hepatocellular carcinoma

    PubMed Central

    TSUNEDOMI, RYOUICHI; IIZUKA, NORIO; YOSHIMURA, KIYOSHI; IIDA, MICHIHISA; TSUTSUI, MASAHITO; HASHIMOTO, NORIAKI; KANEKIYO, SHINSUKE; SAKAMOTO, KAZUHIKO; TAMESA, TAKAO; OKA, MASAAKI

    2013-01-01

    The poor prognosis of hepatocellular carcinoma (HCC) can be explained largely by the high rate of intrahepatic recurrence (IHR). Identification of genes related to IHR is needed to improve the poor prognosis and important for personalized medicine. Eighty-one HCC specimens were used in this study. We screened for IHR-related genes by DNA microarray analysis. The validation of screening was performed by using real-time PCR. The methylation levels in genomic DNAs were measured by quantitative methylation-specific PCR. Six hepatoma cell lines were used for examination of ABCB6 expressional regulation. Time-to-event analyses for recurrence after surgery were analyzed by Kaplan-Meier analysis and Cox regression analysis with cutoff values obtained from receiver operating characteristic (ROC) analysis. We confirmed that ABCB6 mRNA levels were significantly higher in hepatitis C virus (HCV)-related HCCs with early IHR compared to HCV-related HCCs without early IHR (2.5-fold, P=0.01) and the corresponding non-cancerous livers (3.1-fold, P=0.05). Experiments with cell lines showed correlation between DNA methylation and mRNA levels of ABCB6. ROC analysis revealed that mRNA levels (0.81 area under the curve, 88% sensitivity and 72% specificity) and DNA methylation levels (0.81 area under the curve, 80% sensitivity and 80% specificity) of ABCB6 in HCV-related HCCs allowed for the accurate discr