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1

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

PubMed Central

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

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

2010-01-01

2

Image library of biological macromolecules  

Microsoft Academic Search

An Image Library of Biological Macromolecules is described, which contains image and text files related to structures of biological macromolecules. Currently, the Library has ~5000 image files of ~500 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, ~70 DNA structures, 150 proteins and

Jürgen Sühnel

1996-01-01

3

pH-dependent dynamics of complex RNA macromolecules  

PubMed Central

The role of pH-dependent protonation equilibrium in modulating RNA dynamics and function is one of the key unanswered questions in RNA biology. Molecular dynamics (MD) simulations can provide insight into the mechanistic roles of protonated nucleotides, but it is only capable of modeling fixed protonation states and requires prior knowledge of the key residue’s protonation state. Recently, we developed a framework for constant pH molecular dynamics simulations (CPHMDMS?D) of nucleic acids, where the nucleotides’ protonation states are modeled as dynamic variables that are coupled to the structural dynamics of the RNA. In the present study, we demonstrate the application of CPHMDMS?D to the lead-dependent ribozyme; establishing the validity of this approach for modeling complex RNA structures. We show that CPHMDMS?D accurately predicts the direction of the pKa shifts and reproduces experimentally-measured microscopic pKa values with an average unsigned error of 1.3 pKa units. The effects of coupled titration states in RNA structures are modeled, and the importance of conformation sampling is highlighted. The general accuracy of CPHMDMS?D simulations in reproducing pH-dependent observables reported in this work demonstrates that constant pH simulations provides a powerful tool to investigate pH-dependent processes in nucleic acids.

Goh, Garrett B.; Knight, Jennifer L.

2013-01-01

4

The evolutionary transition from RNA to DNA in early cells  

Microsoft Academic Search

Summary The evolution of genetic material can be divided into at least three major phases: first, genomes of “nucleic acid-like” molecules; secondly, genomes of RNA; and finally, double-stranded DNA genomes such as those present in all contemporary cells. Using properties of nucleic acid molecules, we attempt to explain the evolutionary transition from RNA alone as a cellular informational macromolecule prior

A. Lazcano; R. Guerrero; L. Margulis; J. Oró

1988-01-01

5

Long-range macromolecule interaction and “speed reading” long nucleotide sequences in DNA  

NASA Astrophysics Data System (ADS)

Methods based on the phenomenon of the specific long-range interaction between long macromolecules proposed for “speed reading” nucleotide sequences in single DNA molecules. One way is to measure the electric field potential along the preliminary stretched double DNA strand. Another way of information “reading” is to measure deformation of strand elements caused by an electric field that is generated by the “straightening” electrode due to an alternating voltage applied to it. On the base of the obtained information the sequence of nucleotides in the strand could be determined in principle.

Namiot, V. A.; Anashkina, A. A.; Filatov, I. V.; Tumanyan, V. G.; Esipova, N. G.

2013-01-01

6

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

PubMed

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

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

2014-06-28

7

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

PubMed Central

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

Hsu, Tracy; Mitragotri, Samir

2011-01-01

8

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

NASA Astrophysics Data System (ADS)

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

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

9

Linear and ring DNA macromolecules moderately and strongly confined in nanochannels  

NASA Astrophysics Data System (ADS)

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)

Cifra, Peter; Benkova, Zuzana; Bleha, Tomas

2013-03-01

10

RNA-templated DNA origami structures.  

PubMed

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

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

2013-04-11

11

Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription  

PubMed Central

Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3?-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3?-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription.

Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

2007-01-01

12

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

PubMed Central

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

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

2004-01-01

13

Properties of Macromolecules  

NSDL National Science Digital Library

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

Keirle, Matt

2012-07-10

14

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

PubMed Central

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

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

2008-01-01

15

Covalently Linked RNA-DNA Molecule as Initial Product of RNA Tumour Virus DNA Polymerase  

Microsoft Academic Search

Reverse transcriptase from avian myeloblastosis virus initially synthesizes from endogenous RNA template a covalently linked DNA–RNA species, which Verma et al. suggest contains a small RNA primer molecule.

INDER M. VERMA; NORA L. MEUTH; ESTHER BROMFELD; KENNETH F. MANLY; DAVID BALTIMORE

1971-01-01

16

RNA-Linked DNA Fragments In Vitro*  

PubMed Central

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

Sugino, Akio; Okazaki, Reiji

1973-01-01

17

MicroRNA response to DNA damage  

PubMed Central

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution, but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways, composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that microRNAs (miRNAs) play a critical role in regulation of DNA damage response. Here, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.

Wan, Guohui; Mathur, Rohit; Hu, Xiaoxiao; Zhang, Xinna; Lu, Xiongbin

2011-01-01

18

Revisiting RNA-directed DNA methylation  

PubMed Central

RNA-directed DNA methylation (RdDM) involves sequence-specific guiding of the de novo methylation machinery to complementary genomic DNA by RNA molecules. It is still elusive whether guide RNAs bind directly to DNA or to nascent transcripts produced from it. Even the nature of the guide RNAs is not elucidated. RNA interference (RNAi) studies provided a link between RNAi and RdDM indicating that small interfering RNAs (siRNAs) trigger and guide cytosine methylation. The “siRNA hypothesis” is generally accepted. However, recent data demonstrated that RdDM is not always associated with the accumulation of corresponding siRNAs. RdDM triggers may differ from guide RNAs and further studies are needed to clarify if guide RNAs are small or long RNAs, if they are single or double stranded and if they target DNA or nascent transcript.

Dalakouras, Athanasios; Wassenegger, Michael

2013-01-01

19

RNA Splicing Factors and RNA-Directed DNA Methylation  

PubMed Central

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

Huang, Chao-Feng; Zhu, Jian-Kang

2014-01-01

20

Strategies for RNA-Guided DNA Recombination  

NASA Astrophysics Data System (ADS)

We present a model for homologous DNA recombination events guided by double-stranded RNA (dsRNA) templates, and apply this model to DNA rearrangements in some groups of ciliates, such as Stylonychia or Oxytricha. In these organisms, differentiation of a somatic macronucleus from a germline micronucleus involves extensive gene rearrangement, which can be modeled as topological braiding of the DNA, with the template-guided alignment proceeding through DNA branch migration. We show that a graph structure, which we refer to as an assembly graph, containing only 1- and 4-valent vertices can provide a physical representation of the DNA at the time of recombination. With this representation, 4-valent vertices correspond to the alignment of the recombination sites, and we model the actual recombination event as smoothing of these vertices.

Angeleska, Angela; Jonoska, Nataša; Saito, Masahico; Landweber, Laura F.

21

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

PubMed Central

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

Oldenbourg, R; Ruiz, T

1989-01-01

22

Chimeric RNA\\/DNA oligonucleotide-based gene therapy  

Microsoft Academic Search

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

Li-Wen Lai; Yeong-Hau H. Lien

2002-01-01

23

The origin of DNA:RNA hybridization  

Microsoft Academic Search

Conclusions Besides its use in basic research, the DNA:RNA hybridization technique has helped the development of genetic engineering: it is instrumental in the isolation of specific genes that can be inserted into foreign cells, thus modifying their genetic information. Plants, animals, and microorganisms can now be altered to yield improved crops, pest-resistant plants, and a cheaper source of important proteins

Dario Giacomoni

1993-01-01

24

On the early evolution of RNA polymerase  

Microsoft Academic Search

Summary The lines of evidence suggesting that RNA preceded double-stranded DNA as an informational macromolecule are briefly reviewed. RNA polymerase is hypothesized to have been one of the earliest proteins to appear. It is argued that an important vestige of the original enzyme is found in the contemporary eubacterial ?? subunit of DNA-dependent RNA polymerase and its homologues among the

A. Lazcano; J. Fastag; P. Gariglio; C. Ramírez; J. Oró

1988-01-01

25

Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase  

PubMed Central

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ? 8 x 10?3 s?1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5?-adenylylated DNA with a 20-fold lower kcat and a KM ? 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5?-phosphorylated dC or dG residue on the 3? side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

26

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

PubMed Central

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.

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

2013-01-01

27

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

PubMed Central

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

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

2013-01-01

28

DNA Virus MicroRNA and Methods for Inhibiting Same.  

National Technical Information Service (NTIS)

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

S. Pfeffer T. Tuschl

2004-01-01

29

The chemical stability of abasic RNA compared to abasic DNA  

Microsoft Academic Search

We describe the synthesis of an abasic RNA phos- phoramidite carrying a photocleavable 1-(2-nitrophe- nyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 20-O- protecting group together with the analogous DNA and the 20-OMe RNA abasic building blocks. These units were incorporated into RNA-, 20-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards

Pascal A. Kupfer; Christian J. Leumann

2006-01-01

30

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.

31

Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.  

PubMed Central

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described. Images

Axelrod, V D; Vartikyan, R M; Aivazashvili, V A; Beabealashvili, R S

1978-01-01

32

DNA conformation-dependent activities of human mitochondrial RNA polymerase  

Microsoft Academic Search

Mitochondrial RNA polymerase (POLRMT) is a core protein for mitochondrial DNA (mtDNA) transcription. In addition, POLRMT is assumed to be involved in replication, although its exact role is not yet clearly elucidated. We have found novel properties of human POLRMT using a reconstituted transcription system. Various lengths of RNA molecules were synthesized from templates even without a defined promoter sequence,

Atsushi Fukuoh; Kippei Ohgaki; Hinako Hatae; Isao Kuraoka; Yoshimasa Aoki; Takeshi Uchiumi; Howard T. Jacobs; Dongchon Kang

2009-01-01

33

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

SciTech Connect

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

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

1993-09-15

34

Transfer RNA genes of Euglena gracilis chloroplast DNA  

Microsoft Academic Search

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

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

1984-01-01

35

MicroRNA mediates DNA methylation of target genes.  

PubMed

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

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

2014-02-21

36

Size and Base Composition of RNA in Supercoiled Plasmid DNA  

Microsoft Academic Search

The average size and base composition of the covalently integrated RNA segment in supercoiled ColE1 DNA synthesized in Escherichia coli in the presence of chloramphenicol (CM-ColE1 DNA) have been determined by two independent methods. The two approaches yielded similar results, indicating that the RNA segment in CM-ColE1 DNA contains GMP at the 5' end and comprises on the average 25

Peter H. Williams; Herbert W. Boyer; Donald R. Helinski

1973-01-01

37

DNA Virus MicoRNA and Methods for Inhibiting Same.  

National Technical Information Service (NTIS)

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

S. Pfelfer T. H. Tuschl

2004-01-01

38

A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain.  

PubMed Central

We have demonstrated elsewhere that a precisely placed, stalled Escherichia coli RNA polymerase ternary transcription complex (polymerase-RNA-DNA) stays on the DNA template after passage of a DNA replication fork. Moreover, the bypassed complex remains competent to resume elongation of its bound RNA chain. But the simplicity of our experimental system left several important questions unresolved: in particular, might the observation be relevant only to the particular ternary complex that we studied, and can the finding be generalized to a transcribing instead of a stalled RNA polymerase? To address these issues, we have created three additional ternary transcription complexes and examined their fates after passage of a replication fork. In addition, we have examined the fate of moving RNA polymerase molecules during DNA replication. The results suggest that our previous finding applies to all transcription intermediates of the E. coli RNA polymerase. Images

Liu, B; Wong, M L; Alberts, B

1994-01-01

39

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

PubMed Central

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

Rambo, Robert P.; Tainer, John A.

2010-01-01

40

Chemical methods of DNA and RNA fluorescent labeling.  

PubMed Central

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

Proudnikov, D; Mirzabekov, A

1996-01-01

41

Excited states of protonated DNA/RNA bases.  

PubMed

The very fast relaxation of the excited states to the ground state in DNA/RNA bases is a necessary process to ensure the photostability of DNA and its rate is highly sensitive to the tautomeric form of the bases. Protonation of the bases plays a crucial role in many biochemical and mutagenic processes and it can result in alternative tautomeric structures, thus making important the knowledge of the properties of protonated DNA/RNA bases. We report here the photofragmentation spectra of the five protonated DNA/RNA bases. In most of the cases, the spectra exhibit well resolved vibrational structures, with broad bands associated with very short excited state lifetimes. The similarity between the electronic properties, e.g. excitation energy and very short excited state lifetimes for the canonical tautomers of protonated and neutral DNA bases, suggests that the former could also play an important role in the photostability mechanism of DNA. PMID:24752466

Berdakin, Matias; Féraud, Géraldine; Dedonder-Lardeux, Claude; Jouvet, Christophe; Pino, Gustavo A

2014-05-14

42

Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells  

PubMed Central

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.

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

2012-01-01

43

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.  

PubMed

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA. PMID:9358164

Jost, J P; Frémont, M; Siegmann, M; Hofsteenge, J

1997-11-15

44

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

PubMed Central

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

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

2010-01-01

45

DNA computation in mammalian cells: microRNA logic operations.  

PubMed

DNA computation can utilize logic gates as modules to create molecular computers with biological inputs. Modular circuits that recognize nucleic acid inputs through strand hybridization activate computation cascades to produce controlled outputs. This allows for the construction of synthetic circuits that can be interfaced with cellular environments. We have engineered oligonucleotide AND gates to respond to specific microRNA (miRNA) inputs in live mammalian cells. Both single and dual-sensing miRNA-based computation devices were synthesized for the cell-specific identification of endogenous miR-21 and miR-122. A logic gate response was observed with miRNA expression regulators, exhibiting molecular recognition of miRNA profile changes. Nucleic acid logic gates that are functional in a cellular environment and recognize endogenous inputs significantly expand the potential of DNA computation to monitor, image, and respond to cell-specific markers. PMID:23795550

Hemphill, James; Deiters, Alexander

2013-07-17

46

UV protective effects of DNA repair enzymes and RNA lotion.  

PubMed

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes. PMID:18173718

Ke, Malcolm S; Camouse, Melissa M; Swain, Freddie R; Oshtory, Shaheen; Matsui, Mary; Mammone, Thomas; Maes, Daniel; Cooper, Kevin D; Stevens, Seth R; Baron, Elma D

2008-01-01

47

Polyacid macromolecule primers  

DOEpatents

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

Sugama, Toshifumi (Mastic Beach, NY)

1989-01-01

48

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

PubMed Central

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

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

2013-01-01

49

Single Molecule Electrical Sequencing of DNA and RNA  

NASA Astrophysics Data System (ADS)

Gating nanopore devices are composed of nanopores with embedded nanoelectrodes, and they are expected to be one of the core devices used to realize label-free, low-cost DNA sequencing, subsequently leading to 1000-genome sequencing technologies. The operating principle of these nanodevices is based on identifying single base molecules of single DNA passing through a nanopore using a tunneling current between nanoelectrodes. We successfully identified single base molecules of DNA and RNA using tunneling currents. To make gating nanopore devices fit for practical use, core technologies should be integrated on one device chip. One core technology is the identification of single DNA and RNA composed of many base molecules using tunneling currents. We have succeeded in the single-molecule electrical sequencing of DNA and RNA formed by 3 and 7 base molecules, respectively, using a hybrid method of identifying single base molecules via a tunnelling current and random sequencing. A method that controls the speed of a single DNA passing through a nanopore is one core technology that determines the speed and accuracy of sequencing. We successfully developed a method that controls the translocation speed of a single DNA by three orders of magnitude using a voltage between nanoelectrodes.

Taniguchi, Masateru

2013-03-01

50

RNA Splicing: A New Player in the DNA Damage Response  

PubMed Central

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

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

2013-01-01

51

Template-mediated synthesis of lariat RNA and DNA.  

PubMed

Nucleic acid "lariats" have been of great interest to the biological community since their discovery two decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNA introns). We report here the first synthesis of lariat DNA and RNA via template-mediated chemical ligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of any base composition as well as the more biologically relevant lariat RNA. Typically, branched precursors and complementary linear templates ("splints") were dissolved in an equimolar ratio at a total concentration of 10(-4) M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6 buffer. The template-directed cyclization was very efficient, since the amount of circularized lariat product observed in all cases was in the 40-60% range. The lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF mass spectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNA duplexes were fully supportive of the isolated "lasso" structures. Further characterization was conducted by enzymatic degradation with spleen phosphodiesterase (a 3'-exonuclease) and the RNA lariat debranching enzyme, a specific 2',5'-phosphodiesterase. PMID:14575454

Carriero, Sandra; Damha, Masad J

2003-10-31

52

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

PubMed

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

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

2013-01-01

53

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.

54

Photo-Affinity Labeling of tRNA Binding Sites in Macromolecules. I. Linking of the Phenacyl-p-azide of 4-Thiouridine in (Escherichia coli) Valyl-tRNA to 16S RNA at the Ribosomal P Site*  

PubMed Central

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA1Val was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[3H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 Å of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.

Schwartz, Ira; Ofengand, James

1974-01-01

55

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

Microsoft Academic Search

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

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

1998-01-01

56

Cavities in protein-DNA and protein-RNA interfaces.  

PubMed

An analysis of cavities present in protein-DNA and protein-RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein-protein heterocomplexes. With homodimeric proteins protein-DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein-protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein-protein interfaces, protein-DNA interfaces have a higher inclination to be lined by beta-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches. PMID:19494181

Sonavane, Shrihari; Chakrabarti, Pinak

2009-08-01

57

Py-Im Polyamides Distinguish Double Helical DNA and RNA**  

PubMed Central

Groove specificity. Pyrrole-imidazole polyamides are well-known for their specific interactions with the minor groove of DNA. Here we demonstrate that polyamides do not similarly bind duplex RNA, and offer a structural rationale for the molecular-level discrimination of nucleic acid duplexes by minor groove binding ligands.

Chenoweth, David M.; Meier, Jordan L.

2012-01-01

58

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

Microsoft Academic Search

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

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

2005-01-01

59

Simulations Using Random-Generated DNA and RNA Sequences  

ERIC Educational Resources Information Center

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

Bryce, C. F. A.

1977-01-01

60

Visualization of DNA\\/RNA Structure using Temporal CGRs  

Microsoft Academic Search

We present a new DNA\\/RNA sequence visualization technique, Temporal Chaos Game Representation (TCGR), which is an extension of Frequency Chaos Game Representation (FCGR). Unlike earlier CGR variants, TCGR illustrates the distribution of nucleotide sub-patterns at different positions within a sequence, and it can be applied to sets of sequences as well as individual sequences. TCGRs are suitable for short or

Margaret H. Dunham; Donya Quick; Yuhang Wang; Monnie Mcgee; Jim Waddle

2006-01-01

61

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

ERIC Educational Resources Information Center

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

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

2014-01-01

62

Bifacial PNA complexation inhibits enzymatic access to DNA and RNA.  

PubMed

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

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

2014-01-01

63

Analysis of macromolecules, ligands and macromolecule-ligand complexes  

DOEpatents

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

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

2008-12-23

64

A Superfamily of DNA Transposons Targeting Multicopy Small RNA Genes  

PubMed Central

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

Kojima, Kenji K.; Jurka, Jerzy

2013-01-01

65

FUS-regulated RNA metabolism and DNA damage repair  

PubMed Central

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

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

2014-01-01

66

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2?-fluoro-ANA/RNA and DNA/RNA hybrids  

PubMed Central

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2?-fluoro-ANA analog (2?F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2?F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3?-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4?-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2?F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2?F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2?F-ANA/RNA hybrids to elicit RNase H activity.

Denisov, Alexei Yu.; Noronha, Anne M.; Wilds, Christopher J.; Trempe, Jean-Francois; Pon, Richard T.; Gehring, Kalle; Damha, Masad J.

2001-01-01

67

A Course on Macromolecules.  

ERIC Educational Resources Information Center

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

Horta, Arturo

1985-01-01

68

Single-Molecule Electrical Random Resequencing of DNA and RNA  

NASA Astrophysics Data System (ADS)

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

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

2012-07-01

69

The Role of the Largest RNA Polymerase Subunit Lid Element in Preventing the Formation of Extended RNA-DNA Hybrid  

Microsoft Academic Search

Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the “lid”, is formed by an evolutionarily conserved segment of the RNAP largest subunit (?? in bacterial RNAP). The ?? lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated

Tatyana Naryshkina; Konstantin Kuznedelov; Konstantin Severinov

2006-01-01

70

Propensities for loop structures of RNA & DNA backbones.  

PubMed

RNA oligonucleotides exhibit a large tendency to bend and form a loop conformation which is a major motif contributing to their complex three-dimensional structure. This is in contrast to DNA molecules that predominantly form the double-helix structure. In this paper we investigate by molecular dynamics simulation, as well as, by its combination with the replica-exchange method, the propensity of RNA chains containing the GCUAA pentaloop to form spontaneously a hairpin conformation. The results were then compared with those of analogous hybrid oligonucleotides in which the ribose groups in the loop-region were substituted by deoxyriboses. We find that the RNA oligomers exhibit a marginal excess stability to form loop structures. The equilibrium constant for opening the loop to an extended conformation is twice as large in the hybrid than it is in the RNA chain. Analyses of the hydrogen bonds indicate that the excess stability for forming a hairpin is a result of hydrogen bonds the 2'-hydroxyls in the loop region form with other groups in the loop. Of these hydrogen bonds, the most important is the hydrogen bond donated from the 2'-OH at the first position of the loop to N7 of adenine at the forth position. RNA and DNA backbones are characterized by different backbone dihedral angles and sugar puckering that can potentially facilitate or hamper the hydrogen bonds involving the 2'-OH. Nevertheless, the sugar puckerings of all the pentaloop nucleotides were not significantly different between the two chains displaying the C3'-endo conformation characteristic to the A-form double helix. All of the other backbone dihedrals also did not show any considerable difference in the loop-region except of the ?-dihedral. In this case, the RNA loop exhibited bimodal distributions corresponding to, both, the RNA and DNA backbones, whereas the loop of the hybrid chain behaved mostly as that of a DNA backbone. Thus, it is possible that the behavior of the ?-dihedrals in the loop-region of the RNA adopts conformations that facilitate the intra-nucleotide hydrogen bondings of the 2'-hydroxyls, and consequently renders loop structures in RNA more stable. PMID:23933331

Paladino, Antonella; Zangi, Ronen

2013-01-01

71

Rapid methods to extract DNA and RNA from Cryptococcus neoformans.  

PubMed

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations. PMID:12702347

Bolano, A; Stinchi, S; Preziosi, R; Bistoni, F; Allegrucci, M; Baldelli, F; Martini, A; Cardinali, G

2001-12-01

72

RNA DNA discrimination by the antitermination protein NusB.  

PubMed

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2' hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5' and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm. PMID:12662923

Mühlberger, René; Robelek, Rudolf; Eisenreich, Wolfgang; Ettenhuber, Christian; Sinner, Eva Kathrin; Kessler, Horst; Bacher, Adelbert; Richter, Gerald

2003-04-11

73

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

PubMed Central

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

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

1995-01-01

74

Electrically assisted delivery of macromolecules into the corneal epithelium  

PubMed Central

Electrically assisted delivery is noninvasive and has been investigated in a number of ocular drug delivery studies. The objectives of this study were to examine the feasibility of electrically assisted delivery of macromolecules such as small interfering RNA (siRNA) into the corneal epithelium, to optimize the iontophoresis and electroporation methods, and to study the mechanisms of corneal iontophoresis for macromolecules. Anodal and cathodal iontophoresis, electroporation and their combinations were the methods examined with mice in vivo. Cyanine 3 (Cy3) glyceraldehyde 3-phosphate dehydrogenease (GAPDH) siRNA and fluorescein isothiocyanate (FITC)-dextran of different molecular weights (4 to 70 kDa) were the macromolecules studied. Microscopy and histology after cryostat sectioning were used to analyze and compare the delivery of the macromolecules to the cornea. Iontophoresis was effective in delivering siRNA and dextran up to 70 kDa into the cornea. The electroporation method studied was less effective than that of iontophoresis. Although both iontophoresis and electroporation alone can deliver the macromolecules into the cornea, these methods alone were not as effective as the combination of iontophoresis and electroporation (iontophoresis followed by electroporation). The significant enhancement of dextran delivery in anodal iontophoresis suggests that electroosmosis can be a significant flux enhancing mechanism during corneal iontophoresis. These results illustrate the feasibility of electrically assisted delivery of macromolecules such as siRNA into the cornea.

Hao, Jinsong; Li, S. Kevin; Liu, Chia-Yang; Kao, Winston W.Y.

2009-01-01

75

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

Microsoft Academic Search

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration

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

2003-01-01

76

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

NSDL National Science Digital Library

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

2008-10-06

77

Nucleocytoplasmic transport of macromolecules.  

PubMed Central

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

Corbett, A H; Silver, P A

1997-01-01

78

Competitive DNA-RNA Hybridization of Nuclear and Microsomal RNA in Normal, Neoplastic, and Neonatal Liver Tissuel  

Microsoft Academic Search

SUMMARY The nuclear and microsomal RNA of three minimal de viation hepatomas and 10-day-old neonatal rat liver were compared by standard DNA-RNA competitive hybridiza tion techniques. nRNA from 5123C hepatoma and 10-day neonatal liver lack sequences present in adult liver nuclear RNA. These missing sequences appear to be identical in part. nRNA from the slowly growing Morris 9618 hepa toma

Carleton T. Garrett; Robert E. Moore; Carol Katz; Henry C. Pitot

79

HYBRIDIZATION OF RNA FROM ROUS SARCOMA VIRUS WITH CELLULAR AND VIRAL DNA'S  

PubMed Central

The RNA of Rous sarcoma virus (RSV) hybridizes with DNA's isolated from all eukaryotes tested (animal and plant) with the possible exception of yeast. Bacterial DNA's show no homology to RSV-RNA. DNA's from the oncogenic viruses, adenovirus type 12 and SV40 show significant homology with RSV-RNA, but those from nononcogenic adenoviruses types 2 and 4 are not homologous. RSV-RNA hybridized to all of the DNA's examined has the same, or a closely similar, base composition. Moreover, the melting temperatures of all of the hybrids analyzed are very similar, further suggesting that a common base sequence in DNA functions in hybridization with RSV-RNA.

Yoshikawa-Fukada, M.; Ebert, J. D.

1969-01-01

80

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

PubMed Central

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

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

1986-01-01

81

Spectrofluorometric determination of DNA and RNA with berberine  

NASA Astrophysics Data System (ADS)

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

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

1999-08-01

82

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

PubMed Central

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

Pelissier, T; Wassenegger, M

2000-01-01

83

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

PubMed Central

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

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

2013-01-01

84

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

PubMed

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

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

2013-08-01

85

Competitive DNA-RNA Hybridization of Microsomal and Nuclear RNA in Normal Tissues of the Rat1  

Microsoft Academic Search

SUMMARY Purified nuclear and microsomal RNA from rat brain, spleen, kidney, and liver were compared by means of com petitive DNA-RNA hybridization. In the experiments de scribed in this report a close similarity in the sequences de tected by this technique is noted in the nuclear RNA of kidney, brain, and liver, while a portion of these sequences appear to

Carleton T. Garren; Robert E. Moore; Carol Katz; Henry C. Pitot

86

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

PubMed Central

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

Fisk, J. Nick; Newman, Dina L.

2014-01-01

87

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

PubMed Central

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

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

2010-01-01

88

Homology between double-stranded RNA and nuclear DNA of yeast.  

PubMed Central

The relationship between mycoviral double-stranded (ds) RNA and host cell DNA was investigated. Radiolabeled ds RNA was denatured and reannealed in the presence and absence of denatured DNA. RNA from killer strains of the yeast Saccharomyces cerevisiae and from nonkiller derivatives was utilized. The above-mentioned strains, as well as one that lacks all ds RNA, were sources for extracted DNA. Net hybridization of ds RNA to DNA occurred regardless of the strains from which the respective nucleic acids were prepared.

Vodkin, M

1977-01-01

89

Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part A. Optimisation of the assay  

Microsoft Academic Search

SUMMARY: Assay protocols for RNA and DNA in crude plankton extracts using the fluorochrome SYBR Green II are developed here. The method is based on the fluorescence in 3 aliquots: the first measures RNA after DNA digestion; the second measures DNA after RNA digestion; and the third measures residual fluorescence after digestion of both DNA and RNA. This residual fluorescence

ELISA BERDALET; CRISTINA ROLDÁN; M. PILAR OLIVAR; KRISTINE LYSNES

90

Structural analysis of hepatitis C RNA genome using DNA microarrays  

PubMed Central

Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5? non-coding region (5?NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions.

Martell, Maria; Briones, Carlos; de Vicente, Aranzazu; Piron, Maria; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gomez, Jordi

2004-01-01

91

Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers  

SciTech Connect

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

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

2008-11-01

92

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

PubMed

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

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

2014-02-01

93

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

PubMed Central

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

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

2014-01-01

94

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

SciTech Connect

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

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

2010-01-01

95

The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis  

PubMed Central

Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3? end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation.

Berman, Andrea J.; Akiyama, Benjamin M.; Stone, Michael D.; Cech, Thomas R.

2011-01-01

96

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

Microsoft Academic Search

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

N. H. Colburn; R. K. Boutwell

97

Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA  

Microsoft Academic Search

Carboxyl-coated magnetic nanoparticles (MNPs) were used to demonstrate dual functionality: isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled (sc) form of plasmid DNA (pDNA) from agarose gel. These MNPs were attached with 5?-NH2-tagged oligo-(dT)25 primer and were used to isolate mRNA from breast cancer cells. The isolated mRNA was used for amplification of ?-actin to

Tapasree Roy Sarkar; Joseph Irudayaraj

2008-01-01

98

Theoretical studies of DNA-RNA hybrid conformations.  

PubMed Central

Molecular modelling has been used to probe the conformational preferences of double stranded DNA-RNA hybrids. As might be expected, the sugars of the DNA strand have higher conformational flexibility, but, for the majority of the repetitive sequences studied, these sugars prefer a C2-endo pucker, while ribose sugars uniformly adopt a C3-endo pucker. This gives rise to a strongly heteronomous duplex conformation. One exception to this rule involves the thymidine strand of poly(dT).poly(rA), which marginally prefers a C3-endo pucker. Our study further indicates that the DNA strands of the hybrids favour backbone torsions in the canonical B domain, rather than the modified values proposed on the basis of fibre diffraction studies. Backbone conformational transitions can nevertheless be induced leading to an alpha gamma-flip (alpha:gamma, g-/g(+)-->t/t) or to the alpha beta gamma-flip form proposed from fibre studies (alpha:beta:gamma, g-/t/g(+)-->t/g+/t). The latter transition is also found to be linked to BI-->BII transitions (epsilon:zeta, t/g(-)-->g-/t). Images

Sanghani, S R; Lavery, R

1994-01-01

99

Theoretical studies of DNA-RNA hybrid conformations.  

PubMed

Molecular modelling has been used to probe the conformational preferences of double stranded DNA-RNA hybrids. As might be expected, the sugars of the DNA strand have higher conformational flexibility, but, for the majority of the repetitive sequences studied, these sugars prefer a C2-endo pucker, while ribose sugars uniformly adopt a C3-endo pucker. This gives rise to a strongly heteronomous duplex conformation. One exception to this rule involves the thymidine strand of poly(dT).poly(rA), which marginally prefers a C3-endo pucker. Our study further indicates that the DNA strands of the hybrids favour backbone torsions in the canonical B domain, rather than the modified values proposed on the basis of fibre diffraction studies. Backbone conformational transitions can nevertheless be induced leading to an alpha gamma-flip (alpha:gamma, g-/g(+)-->t/t) or to the alpha beta gamma-flip form proposed from fibre studies (alpha:beta:gamma, g-/t/g(+)-->t/g+/t). The latter transition is also found to be linked to BI-->BII transitions (epsilon:zeta, t/g(-)-->g-/t). PMID:7514787

Sanghani, S R; Lavery, R

1994-04-25

100

RNA 3?-Phosphate Cyclase (RtcA) Catalyzes Ligase-like Adenylylation of DNA and RNA 5?-Monophosphate Ends*  

PubMed Central

RNA 3?-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2?,3?-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PPi; transfer of AMP from Rtc to an RNA 3?-phosphate to form an RNA(3?)pp(5?)A intermediate; and attack by the terminal nucleoside O2? on the 3?-phosphate to form an RNA 2?,3?-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5?-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5?-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5? modification reaction requires the His309 nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [32P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5?-phosphate at a 5?-PO4/3?-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3?-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5?-adenylyltransferase of RtcA might play a role.

Chakravarty, Anupam K.; Shuman, Stewart

2011-01-01

101

Analysis of Loss of Nuclear RNA in Azo Dye-induced Hepatoma by DNA-RNA Competitive Hybridization1  

Microsoft Academic Search

DNA-RNA hybridization studies, using nuclear RNA's (nRNA's) labeled in vivo and in vitro with high specific radioac tivities, were performed to compare the nRNA populations of normal rat liver, livers treated with 3'-methyl-4-dimethylamino- azobenzene (3'-Me-DAB), and 3'-Me-DAB-induced hepato- mas. The study with normal liver nRNA labeled by i.p. injection of (3H)orotic acid indicated that the nuclei of a 3'-Me-DAB- induced

Mitsutaro Akao; Keiko Kuroda

1981-01-01

102

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

PubMed

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

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

2013-10-30

103

Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA  

PubMed Central

In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.

Dell'Anno, A.; Fabiano, M.; Duineveld, G. C. A.; Kok, A.; Danovaro, R.

1998-01-01

104

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

PubMed Central

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

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

2014-01-01

105

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.  

PubMed Central

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging. Images

Valpuesta, J M; Donate, L E; Mier, C; Herranz, L; Carrascosa, J L

1993-01-01

106

Anchoring Nascent RNA to the DNA Template Could Interfere with Transcription  

PubMed Central

During normal transcription, the nascent RNA product is released from the DNA template. However, in some cases, the RNA remains bound or can become reattached to the template DNA duplex (for example, through R-loop formation). We have analyzed the effect on transcription elongation of nascent RNA anchoring to the template DNA duplex. Because the RNA polymerase follows a helical path along DNA duplex during transcription, the anchoring would result in wrapping the nascent RNA around the DNA in the region between the anchoring point and the translocating polymerase. This wrapping would cause an unfavorable loss of conformation entropy of the nascent RNA. It consequently would create an apparent force to unwrap the RNA by disrupting either the transcription complex or the anchoring structure. We have estimated that this force would be comparable to those required to melt nucleic acid duplexes or to arrest transcription elongation in single-molecule experiments. We predict that this force would create negative supercoiling in the DNA duplex region between the anchoring point and the transcribing RNA polymerase: this can promote the formation of unusual DNA structures and facilitate RNA invasion into the DNA duplex. Potential biological consequences of these effects are discussed.

Belotserkovskii, Boris P.; Hanawalt, Philip C.

2011-01-01

107

Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection.  

PubMed

Multiplexed detection of oligonucleotide targets has been performed with gold nanoparticle probes labeled with oligonucleotides and Raman-active dyes. The gold nanoparticles facilitate the formation of a silver coating that acts as a surface-enhanced Raman scattering promoter for the dye-labeled particles that have been captured by target molecules and an underlying chip in microarray format. The strategy provides the high-sensitivity and high-selectivity attributes of gray-scale scanometric detection but adds multiplexing and ratioing capabilities because a very large number of probes can be designed based on the concept of using a Raman tag as a narrow-band spectroscopic fingerprint. Six dissimilar DNA targets with six Raman-labeled nanoparticle probes were distinguished, as well as two RNA targets with single nucleotide polymorphisms. The current unoptimized detection limit of this method is 20 femtomolar. PMID:12202825

Cao, YunWei Charles; Jin, Rongchao; Mirkin, Chad A

2002-08-30

108

Configurational diffusion of coal macromolecules  

SciTech Connect

The objective of this project is to investigate the phenomenon of hindered diffusion of coal macromolecules in idealized porous media. Tasks towards this objective include: Construct a diffusion cell with ideal pore structure for determination of diffusion coefficients, prepare and characterize ideal porous membranes, perform model compound experiments to calibrate and test diffusion apparatus and methodology, prepare and characterize coal macromolecules, and analyze data to evaluate the diffusional behavior of coal macromolecules. This report describes work on the hindered diffusion of tetraphenylporphine and asphaltene. 18 refs., 3 figs., 4 tabs.

Guin, J.A.; Curtis, C.W.; Tarrer, A.R.

1990-01-01

109

The Tree of Life's Macromolecules  

NSDL National Science Digital Library

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

Project, Molecular L.

110

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

Microsoft Academic Search

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

J. M. Amabis; Dulce Cabral

1970-01-01

111

Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet  

Microsoft Academic Search

A new Watson-Crick base pair, with a hydrogen bonding pattern different from that in the A.T and G.C base pairs, is incorporated into duplex DNA and RNA by DNA and RNA polymerases and expands the genetic alphabet from 4 to 6 letters. This expansion could lead to RNAs with greater diversity in functional groups and greater catalytic potential.

Joseph A. Piccirilli; Steven A. Benner; Tilman Krauch; Simon E. Moroney

1990-01-01

112

Cytological localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera  

Microsoft Academic Search

Molecular hybridization of radioactive ribosomal RNA with the DNA of cytological preparations is used to study the distribution of the ribosomal cistrons within the polytene chromosomes of three species of Diptera. It is shown that in Drosophila hydei the ribosomal cistrons are located within the nucleolus. In Rhynchosciara hollaenderi and Sciara coprophila, DNA coding for ribosomal RNA is present both

Mary Lou Pardue; Susan A. Gerbi; Ronald A. Eckhardt; Joseph G. Gall

1970-01-01

113

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

PubMed Central

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

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

2014-01-01

114

miRNA Gene Promoters Are Frequent Targets of Aberrant DNA Methylation in Human Breast Cancer  

PubMed Central

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

Vrba, Lukas; Munoz-Rodriguez, Jose L.; Stampfer, Martha R.; Futscher, Bernard W.

2013-01-01

115

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

PubMed Central

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

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

2012-01-01

116

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

PubMed Central

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

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

2013-01-01

117

An attempt to detect siRNA-mediated genomic DNA modification by artificially induced mismatch siRNA in Arabidopsis.  

PubMed

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

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

2013-01-01

118

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.  

PubMed Central

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

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

1995-01-01

119

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

PubMed Central

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

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

1966-01-01

120

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

NASA Technical Reports Server (NTRS)

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

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

1982-01-01

121

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

SciTech Connect

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

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

2006-01-01

122

RNA-directed DNA polymerase activity of reticuloendotheliosis virus: characterization of the endogenous and exogenous reactions.  

PubMed Central

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.

Waite, M R; Allen, P T

1975-01-01

123

Use of RNA/DNA chimeric primers for improved nucleic acid amplification reactions  

US Patent & Trademark Office Database

Methods are provided for amplification of a nucleic acid sequence. The method use RNA/DNA chimeric oligonucleotides as primers. The primers have RNA residues scattered along their length and no two ribonucleotides in the prime are adjacent to one another. The methods are useful for reducing non-specific amplification products, such as primer dimers. The invention also provides kits comprising RNA/DNA chimeric oligonucleotide primers for practicing the amplification methods.

2013-06-11

124

Delivery of shRNA using gold nanoparticle–DNA oligonucleotide conjugates as a universal carrier  

Microsoft Academic Search

The efficient delivery of nucleic acids into mammalian cells is a central aspect of research involving cell biology and medical applications, including the clinical treatment of genetic disorders. We report an efficient small hairpin RNA (shRNA) delivery system that utilizes a single species of gold nanoparticle–DNA oligonucleotide conjugate (AuNP–DNA oligo) as a universal carrier. In vitro synthesized shRNA that is

Sang-Mi Ryou; Sudeok Kim; Hyun Hye Jang; Jae-Hong Kim; Ji-Hyun Yeom; Min Sik Eom; Jeehyeon Bae; Min Su Han; Kangseok Lee

2010-01-01

125

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

SciTech Connect

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

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

2011-07-25

126

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

PubMed

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

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

2013-01-14

127

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

PubMed

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

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

2013-01-01

128

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

PubMed Central

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

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

2013-01-01

129

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

PubMed

Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV-ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups. PMID:9714252

Hu, C C; Sanger, M; Ghabrial, S A

1998-08-01

130

Architectural Arrangement of the Small Nuclear RNA (snRNA)-activating Protein Complex 190 Subunit (SNAP190) on U1 snRNA Gene Promoter DNA*  

PubMed Central

Myb repeats ?52 amino acid residues in length were first characterized in the oncogenic Myb transcription factor, which contains three tandem Myb repeats in its DNA-binding domain. Proteins of this family normally contain either one, two, or three tandem Myb repeats that are involved in protein-DNA interactions. The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes. This complex binds to an essential promoter element, the proximal sequence element, centered ?50 base pairs upstream of the transcription start site of snRNA genes. SNAP190, the largest subunit of SNAPc, uncharacteristically contains 4.5 tandem Myb repeats. Little is known about the arrangement of the Myb repeats in the SNAPc-DNA complex, and it has not been clear whether all 4.5 Myb repeats contact the DNA. By using a site-specific protein-DNA photo-cross-linking assay, we have now mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. The results reveal the topological arrangement of the 4.5 SNAP190 Myb repeats relative to the DNA and to each other when SNAP190 is bound to a U1 promoter as a subunit of SNAPc.

Doherty, Matthew T.; Kang, Yoon Soon; Lee, Cheryn; Stumph, William E.

2012-01-01

131

RNA Synthesis Inhibitors Alter the Subnuclear Distribution of DNA Topoisomerase I1  

Microsoft Academic Search

The acute effect of RNA and DNA synthesis inhibitors on DNA topoi- somerase (topo) I localization within cells was examined. Indirect imimi- nofluorescence revealed that topo I was distributed throughout the nuclei hut was concentrated in nucleoli of untreated K562 leukemia cells and A549 non-small cell lung cancer cells. Treatment with the DNA polymer- ase inhibitor aphidicolin did not alter

Christopher A. Buckwalter; Amy H. Lin; Akihiko Tanizawa; Yves G. Pommier; Yung-Chi Cheng; Scott H. Kaufmann

132

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.  

PubMed

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation. PMID:21766868

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

133

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

PubMed

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

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

2009-06-01

134

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

PubMed Central

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

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

2013-01-01

135

Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA  

Microsoft Academic Search

We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene

P. Cantatore; M. Roberti; G. Rainaldi; C. Saccone; M. N. Gadaleta

1988-01-01

136

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

PubMed Central

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

Tan, Siun Chee; Yiap, Beow Chin

2009-01-01

137

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

PubMed

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

Gasiunas, Giedrius; Siksnys, Virginijus

2013-11-01

138

Structure and biology of cartilage and bone matrix noncollagenous macromolecules  

Microsoft Academic Search

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

DICK HEINEGARD; AKE OLDBERG

139

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions  

Microsoft Academic Search

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes

David W. Deamer; Gail L. Barchfeld

1982-01-01

140

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3?-to-5? DNA Translocase and Helicase That Prefers to Unwind 3?-Tailed RNA:DNA Hybrids*  

PubMed Central

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

Ordonez, Heather; Shuman, Stewart

2013-01-01

141

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

Microsoft Academic Search

Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with

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

2008-01-01

142

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

PubMed

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

Kim, Eunhye; Paeng, Insook Rhee

2014-01-01

143

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

PubMed

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

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

2014-05-01

144

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

PubMed Central

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

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

2014-01-01

145

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

PubMed

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

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

2014-01-01

146

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

PubMed

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

Zhang, Hui; Guo, Peixuan

2014-05-15

147

Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA  

PubMed Central

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

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

1974-01-01

148

Novel stem-loop probe DNA arrays: detection of specific acetotrophic 16S ribosomal RNA signatures.  

PubMed

In a recent study, we showed how novel stem-loop DNA probes on dendron-modified aldehyde substrates could be used to detect synthetic nucleic acid targets without amplification. In this article, we demonstrate the application of stem-loop DNA probes as arrays for the detection of specific families and genera of methane-producing bacteria from sludge samples harvested from an anaerobic digester using 16S ribosomal RNA (rRNA) signatures. Specific 16S rRNA could be detected in samples that had 0.2ng/?l total sludge RNA without any target amplification. PMID:23256923

Boateng, Jonas; Zahorchak, Robert; Peek, Joel; Chittur, Krishnan

2013-04-01

149

Small RNA molecules related to the Alu family of repetitive DNA sequences.  

PubMed Central

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA. Images

Leinwand, L A; Wydro, R M; Nadal-Ginard, B

1982-01-01

150

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

PubMed Central

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.

Woo, Hye Ryun; Richards, Eric J

2008-01-01

151

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation  

PubMed Central

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 was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development.

Thilly, William G; Gostjeva, Elena V; Koledova, Vera V; Zukerberg, Lawrence R; Chung, Daniel; Fomina, Janna N; Darroudi, Firouz; Stollar, B David

2014-01-01

152

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA  

PubMed Central

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3?UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT–qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Lopez de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, Maria; Urdinguio, Rocio G.; Fraga, Mario F.; Jacinto, Filipe V.; Esteller, Manel

2009-01-01

153

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

PubMed Central

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

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

2011-01-01

154

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

PubMed Central

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.

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

155

Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA and rRNA-Based Microbial Community Composition  

Microsoft Academic Search

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription- PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons

ROBERT I. GRIFFITHS; ANDREW S. WHITELEY; ANTHONY G. O'DONNELL; MARK J. BAILEY

2000-01-01

156

Detection of messenger RNA from the isoleucine-valine operons of Salmonella typhimurium by heterologous DNA-RNA hybridization: Involvement of transfer RNA in transcriptional repression  

Microsoft Academic Search

A hybridization assay using Escherichia coli K-12 DNA isolated from the specialized transducing bacteriophage ?CI857St68h80 dilv was used to examine the rate of synthesis of the messenger RNA's (mRNA) derived from the isoleucine-valine (ilv) gene cluster of Salmonella typhimurium. In all cases examined, changes in ilv enzyme levels could be correlated with changes in the rate of synthesis of ilv

Geoffrey Childs; Frank Sonnenberg; Martin Freundlich

1977-01-01

157

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

PubMed

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

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

2013-05-01

158

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...

159

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

ERIC Educational Resources Information Center

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

Godsell, P. M.; Balls, M.

1973-01-01

160

Interactions of trichloroethylene with DNA in vitro and with RNA and DNA of various mouse tissues in vivo.  

PubMed

The covalent binding of 14C-1,1,2-trichloroethylene (14C-TRI) metabolites to calf thymus DNA in vitro and to RNA and DNA of mouse brain, lung, liver, kidney, spleen, pancreas, and testis after repeated i.p. injections has been studied. Hydrolysates of DNA reacted with 14C-TRI in vitro and hydrolysates of RNA and DNA from selected organs were separated on Aminex A6 for quantitation of alkylation products. The presence of 3,N4-etheno(deoxy)cytidine, 1,N6-etheno(deoxy)adenosine and 1,N6-ethenoadenine was investigated. No radioactivity could be registered in DNA incubated with 14C-TRI in the absence of liver microsomes. Covalent binding of 14C-TRI to DNA took place in the presence of liver microsomes from control mice. The binding was enhanced by 50% if liver microsomes from phenobarbital pretreated mice were used. The radioactivity in DNA reacted with 14C-TRI and microsomes from control mice was eluted in early fractions and together with thymidine. The same two peaks appeared on chromatography of DNA incubated with 14C-TRI and liver microsomes from phenobarbital pretreated mice. In addition, radioactivity was eluted together with 1,N6-ethenoadenine. Radioactivity was registered in RNA and DNA from all of the studied organs after i.p. injections of 14C-TRI. The radioactivity in RNA increased in the order brain less than testis less than pancreas less than kidney less than liver less than lung less than spleen. The radioactivity in DNA increased in the order brain less than kidney less than testis less than lung less than pancreas less than liver less than spleen. Aminex A6 chromatography revealed that the entire radioactivity in RNA from liver and kidney and in DNA from kidney, testis, lung, pancreas, and spleen was due to metabolic incorporation, particularly into guanine and adenine. This finding indicates that the C-C bond in TRI is split, with the formation of C1-fragments, during biotransformation in vivo. In liver DNA, the metabolic incorporation of radioactivity was insignificant. Instead, the dominant part of the radioactivity in liver DNA was eluted in early fractions. The elution profile of radioactivity in liver DNA gave no direct evidence of the formation of TRI-DNA adducts in vivo. No etheno-derivatives were identified as alkylation products of TRI in vivo, which is consistent with current theories of the metabolic fate of TRI. PMID:6197950

Bergman, K

1983-11-01

161

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres*  

PubMed Central

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

Xu, Yan; Ishizuka, Takumi; Yang, Jie; Ito, Kenichiro; Katada, Hitoshi; Komiyama, Makoto; Hayashi, Tetsuya

2012-01-01

162

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

PubMed Central

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

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

2009-01-01

163

Targeted Delivery of siRNA-Generating DNA Nanocassettes Using Multifunctional Nanoparticles  

PubMed Central

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

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

2013-01-01

164

Macromolecules in Undergraduate Physical Chemistry.  

ERIC Educational Resources Information Center

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

Mattice, Wayne L.

1981-01-01

165

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

PubMed

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

Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia

2013-09-01

166

Carbon Catabolism and Synthesis of Macromolecules During Spore Germination of Microsporum gypseum1  

PubMed Central

Either d- or l-leucine (10?3m) and unsaturated long-chain fatty acids such as oleic, linoleic, and arachidonic (10?4m) significantly stimulated macroconidia germination of Microsporum gypseum. Saturated long-chain fatty acids did not affect germination, whereas saturated short-chain fatty acids such as caprylic, hexanoic, and butyric were completely inhibitory. Germination was followed by an increase in endogenous respiration and a decrease in dry weight of approximately 5% at 4 hr. Endogenous fatty acids and soluble carbohydrates were utilized significantly during germination. Tritiated leucine, uridine, and thymidine were incorporated respectively into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) fractions within the first 5 min of germination. Incorporation of oleic-1-C14 into RNA and protein was significantly increased after germ tube development. Net synthesis of RNA and protein started prior to germ tube protrusion. Increase in DNA could be detected only later. A significant increase in RNA and protein during the 4th hr of germination was correlated with vegetative development. Inhibition of respiration and incorporation of leucine-H3 and uridine-H3 into corresponding macromolecules by dl-fluorophenylalanine and phenethyl alcohol started before germ tube appearance. Griseofulvin significantly inhibited incorporation of uridine-H3 and thymidine-H3, but not of leucine-H3. This inhibition occurred only after initial vegetative development. In contrast to the two other inhibitors, which substantially inhibited germination, griseofulvin only slightly retarded the period of germination and did not affect respiration.

Barash, I.; Conway, M. Loretto; Howard, D. H.

1967-01-01

167

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 ...

168

Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template.  

PubMed Central

A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template. Images

Donahue, B A; Yin, S; Taylor, J S; Reines, D; Hanawalt, P C

1994-01-01

169

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

PubMed Central

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

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

2012-01-01

170

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

PubMed Central

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

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

2012-01-01

171

I N SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA  

Microsoft Academic Search

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chro- mosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2) . Salivary glands were incubated with tritiated nucleosides . The labeled RNA was extracted from microdissected nuclei and hybridized to denatured

B. LAMBERT; L. WIESLANDER; B. DANEHOLT; E. EGYHAZI; U. RINGBORG

1972-01-01

172

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants.  

PubMed Central

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

Wang, M B; Wesley, S V; Finnegan, E J; Smith, N A; Waterhouse, P M

2001-01-01

173

DNA-dependent RNA polymerases from Schneider 2-L cells of Drosophila . I. Preliminary characterization  

Microsoft Academic Search

The DNA-dependent RNA polymerases of Schneider 2-L cells of Drosophila melanogaster are described. These cells contain five readily detectable forms of this enzyme, polymerases Ia, Ib, IIIa, II, and IIIb, which elute from DEAE-Sephadex at 0.08, 0.12, 0.15, 0.20, and 0.22 m ammonium sulfate, respectively. RNA polymerases IIIa and IIIb, which each constitute about 5–10% of the total RNA polymerase

John P. Phillips; Donald G. Somers; C. Feng

1982-01-01

174

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

Microsoft Academic Search

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

Hye Ryun Woo; Eric J Richards

2008-01-01

175

Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases*  

PubMed Central

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

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

2012-01-01

176

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction.  

PubMed

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding. PMID:17196678

Lee, Hunjoong; Diavatis, Theodore; Tennakoon, Sanka; Yu, Peilin; Gao, Xiaolian

2007-01-01

177

Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site.  

PubMed

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site. PMID:17526498

Kashkina, Ekaterina; Anikin, Michael; Brueckner, Florian; Lehmann, Elisabeth; Kochetkov, Sergey N; McAllister, William T; Cramer, Patrick; Temiakov, Dmitry

2007-07-27

178

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

PubMed Central

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

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

2011-01-01

179

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

PubMed Central

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

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

2012-01-01

180

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.  

PubMed

MicroRNAs (miRNAs) are small regulatory RNAs (size ?21nt to ?25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method. PMID:24616905

Shah, Pratik; Thulstrup, Peter Waaben; Cho, Seok Keun; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Bjerrum, Morten Jannik; Yang, Seong Wook

2014-05-01

181

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

SciTech Connect

Studies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and malignant bone neoplasms. Benign tumors manifested predominantly DNA diploidy and low proliferative activity, whereas the majority of malignant tumors were DNA aneuploid and showed high proliferation rate. No significant difference in the RNA content between different histopathologic categories was found. We observed, however, a distinct and consistently high RNA content pattern in giant cell tumors, aneurysmal bone cysts, and chondroblastomas that may be useful in their differential diagnosis. Analysis of different prognostic factors in malignant tumors indicated that histologic grade and DNA content are significant prognostic factors. Further analysis of malignant tumors showed that a correlation between the proliferative activity and the clinical outcome in the low grade category and between RNA content and patients` survival in osteosarcomas. Our study also showed that preoperative treatment significantly impacted on the extent of the proliferative fraction in malignant tumors. We conclude that DNA/RNA analysis of bone tumor may assist in: (1) the differential diagnosis of certain bone tumors, (2) evaluation of treatment response, and (3) the biological assessment of osteosarcomas. 38 refs., 4 figs., 5 tabs.

El-Naggar, A.K.; Hurr, K.; Tu, Z.N.; Teague, K.; Raymond, K.A.; Ayala, A.G.; Murray, J. [Univ. of Texas, Houston, TX (United States)

1995-03-01

182

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

PubMed Central

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

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

2013-01-01

183

RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients  

PubMed Central

Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme- containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[- 3H]DNA hybridization.

1975-01-01

184

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

SciTech Connect

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

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

1992-07-01

185

The chemical structure of DNA sequence signals for RNA transcription  

NASA Technical Reports Server (NTRS)

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

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

1982-01-01

186

Heat shock protein-mediated cell penetration and cytosolic delivery of macromolecules by a telomerase-derived peptide vaccine.  

PubMed

A reverse-transcriptase-subunit of telomerase (hTERT) derived peptide, GV1001, has been developed as a vaccine against various cancers. Here, we report an unexpected function of GV1001 as a cell-penetrating peptide (CPP). GV1001 was delivered into a variety of cells including various cancer cell lines and primary blood cells. Moreover, the delivered GV1001 was predominantly located in the cytoplasm of the cells, while a significantly higher proportion of TAT peptide was localized in the nucleus. Macromolecules such as proteins, DNA and siRNA, which were linked to GV1001 by direct covalent conjugation or non-covalent complexation through poly-lysine, were successfully delivered into cells, indicating that GV1001 can be used as a carrier for macromolecules. Expression of the delivered DNA, and lowered expression of the target gene by the delivered siRNA, suggest the potential therapeutic use of GV1001. Pull-down analysis identified Heat Shock Protein 90 (HSP90) and 70 (HSP70) as GV1001 interacting proteins. Treatment of Anti-HSP90 and HSP70 antibodies lowered the internalization of GV1001, indicating that the interaction is critical for the efficient internalization of GV1001. Collectively, the results of this study suggest the pharmaceutical potential of GV1001, already proven safe in clinical trials, as a carrier for the delivery of macromolecular therapeutics into cells, in addition to its own anti-cancer activity. PMID:23827187

Lee, Seoung-Ae; Kim, Bo-Ram; Kim, Bu-Kyung; Kim, Dong-Won; Shon, Won-Jun; Lee, Na-Rae; Inn, Kyung-Soo; Kim, Bum-Joon

2013-10-01

187

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 ...

188

The influence of RNA integrity, purity and cDNA labelling on glass slide cDNA microarray image quality  

PubMed Central

Background The accuracy of gene expression measurements generated using cDNA microarrays is dependent on the quality of the image generated following hybridization of fluorescently labelled cDNA. It is not known how this image is influenced by sample preparation factors which such as RNA quality, cDNA synthesis and labelling efficiency. Results In this study we used a simple metric based on the ratio of the total feature (F) and background (B) fluorescence, which correlates with the visual assessment of 60 microarray images, to determine the influence of sample preparation on image quality. Results indicate that RNA purity (A260/A280) and integrity (18S:28S ratio) do not strongly influence microarray image quality. cDNA having an nucleotide to dye ratio greater than 100 produced poor microarray images, however, cDNA labelled more efficiently was not a guarantee of a better image. The data also indicate that the array image quality is not improved by loading more cDNA into the hybridization mixture however poor image quality did result from a disproportionate amounts of Cy5 and Cy3 labelled cDNA. Conclusion This study provides insight into the source of variation in microarray image analysis introduced during sample preparation and will assist in the standardisation of cDNA glass slide microarray protocols.

2004-01-01

189

Viral RNA and DNA trigger common antiviral responses in mesangial cells.  

PubMed

Extrarenal viral infections commonly trigger glomerulonephritis, usually in association with immune complex disease. The Ig component of immune complexes can activate glomerular cell Fc receptors, but whether complexed viral nucleic acids contribute to glomerular inflammation remains unknown. Because of the types of Toll-like receptors (Tlrs) expressed by glomerular mesangial cells, we hypothesized that viral single-stranded RNA and DNA would activate mesangial cells via Tlr-independent pathways and trigger overlapping antiviral immune responses. Consistent with this hypothesis, 5'-triphosphate RNA (3P-RNA) and non-CpG DNA activated murine primary glomerular mesangial cells to secrete Cxcl10 and Il-6 even in cells derived from mice deficient in the Tlr adaptor proteins Myd88 and Trif. Transcriptome analysis revealed that 3P-RNA and non-CpG-DNA triggered almost identical gene expression programs, especially the proinflammatory cytokine Il-6, several chemokines, and genes related to type I IFN. We observed similar findings in glomerular preparations after injecting 3P-RNA and non-CpG-DNA in vivo. These effects depended on the formation of complexes with cationic lipids, which enhanced nucleic acid uptake into the cytosol of mesangial cells. Small interfering RNA studies revealed that 3P-RNA recognition involves Rig-1, whereas non-CpG-DNA did not require Rig-1 or Dai to activate glomerular mesangial cells. We conclude that 3P-RNA and double-stranded DNA trigger a common, TLR-independent, antiviral response in glomerular mesangial cells, which may promote glomerulonephritis in the setting of viral infection. PMID:19713315

Allam, Ramanjaneyulu; Lichtnekert, Julia; Moll, Anton G; Taubitz, Anela; Vielhauer, Volker; Anders, Hans-Joachim

2009-09-01

190

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

PubMed Central

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

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

2013-01-01

191

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

NASA Astrophysics Data System (ADS)

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

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

2014-01-01

192

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

PubMed Central

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

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

2013-01-01

193

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

PubMed Central

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

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

2014-01-01

194

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

PubMed

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

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

2014-02-01

195

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

PubMed Central

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

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

2011-01-01

196

Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells  

PubMed Central

A strategy is presented for the live cell imaging of messenger RNA (mRNA) using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNAse degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells, and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently-stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.

Jayagopal, Ashwath; Halfpenny, Kristin C.; Perez, Jonas W.; Wright, David W.

2010-01-01

197

An Rrp6-like Protein Positively Regulates Noncoding RNA Levels and DNA Methylation in Arabidopsis.  

PubMed

Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through noncoding RNA degradation and mRNA quality control, including exosomal RNA decay and transcript retention triggered by defective RNA processing. It is unclear whether Rrp6 can positively regulate noncoding RNAs and whether RNA retention occurs in normal cells. Here we report that AtRRP6L1, an Arabidopsis Rrp6-like protein, controls RNA-directed DNA methylation through positive regulation of noncoding RNAs. Discovered in a forward genetic screen, AtRRP6L1 mutations decrease DNA methylation independently of exosomal RNA degradation. Accumulation of Pol V-transcribed scaffold RNAs requires AtRRP6L1 that binds to RNAs in vitro and in vivo. AtRRP6L1 helps retain Pol V-transcribed RNAs in chromatin to enable their scaffold function. In addition, AtRRP6L1 is required for genome-wide Pol IV-dependent siRNA production that may involve retention of Pol IV transcripts. Our results suggest that AtRRP6L1 functions in epigenetic regulation by helping with the retention of noncoding RNAs in normal cells. PMID:24726328

Zhang, Huiming; Tang, Kai; Qian, Weiqiang; Duan, Cheng-Guo; Wang, Bangshing; Zhang, Heng; Wang, Pengcheng; Zhu, Xiaohong; Lang, Zhaobo; Yang, Yu; Zhu, Jian-Kang

2014-05-01

198

RNase-like domain in DNA-directed RNA polymerase II.  

PubMed Central

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function. Images

Shirai, T; Go, M

1991-01-01

199

Particles with RNA of High Molecular Weight and RNA-Directed DNA Polymerase in Human Brain Tumors  

PubMed Central

We have previously shown that neoplastic cells of human breast cancers, leukemias, lymphomas, and sarcomas contain particles similar to the viruses that have been established as etiologic agents of these diseases in mice. The present paper concerns tumors of the central nervous system for which no suitable animal model or corresponding virus exists. Nevertheless, using the simultaneous detection test, we showed that human brain tumors contain 70S RNA and RNA-directed DNA polymerase encapsulated in a particulate component possessing a density of 1.17 g/ml. These particles satisfy the three diagnostic criteria that characterize RNA tumor viruses of animals. 24 Out of 26 (92%) of the most malignant (glioblastoma and medulloblastoma) brain tumors examined contained these virus-like entities.

Cuatico, W.; Cho, J.-R.; Spiegelman, S.

1973-01-01

200

Automated serial extraction of DNA and RNA from biobanked tissue specimens  

PubMed Central

Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage.

2013-01-01

201

Novel DNA-binding properties of the RNA-binding protein TIAR  

PubMed Central

TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR–RNP complex formation with IL-8 and VEGF 3?-untranslated regions (3?-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3?-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA (Kdapp=1.6×10?9M versus 9.4 × 10?9 M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands.

Suswam, Esther A.; Li, Yan Yan; Mahtani, Harry; King, Peter H.

2005-01-01

202

Rapamycin inhibits pre-B acute lymphoblastic leukemia cells by downregulating DNA and RNA polymerases.  

PubMed

Rapamycin has been shown to inhibit the growth of leukemic cells via an unknown mechanism. In our current study, we show that rapamycin activates autophagy in pediatric t(1;19) pre-B acute lymphoblastic leukemia (pre-B ALL) cells and thereby inhibits proliferation and induces growth arrest in these cells. Rapamycin was found to downregulate an extensive array of positive cell cycle regulators, reduce the total DNA and RNA levels, and specifically downregulate the gene transcription of DNA pol ?1 and RNA pol II. Furthermore, we show that both rapamycin and starvation caused a downregulation of the DNA pol ?1 and RNA pol II proteins which was reversed by the autophagy inhibitor 3-MA. Consistent with the results of our autophagic flux analysis, confocal microscopy indicated that both rapamycin and starvation cause the colocalization of DNA pol ?1 and RNA pol II with GFP-LC3 at autophagosomes. This colocalization was blocked by the autophagy inhibitor bafilomycin A1 which inhibits the fusion between autophagosomes and lysosomes. These data suggest that rapamycin inhibits the growth of pediatric t(1;19) pre-B ALL cells through both transcriptional inhibition and autophagic degradation of DNA pol ?1 and RNA pol II. PMID:24939216

Wang, Zhijian; Xu, Fei; Yuan, Na; Niu, Yuna; Lin, Weiwei; Cao, Yan; Cai, Jinyang; Song, Lin; Li, Xin; Fang, Yixuan; Zhao, Wenli; Hu, Shaoyan; Chen, Suning; Zhang, Suping; Wang, Jianrong

2014-08-01

203

Structure and Stability of Individual DNA or RNA Hairpin Molecules Captured in an Ion Channel  

NASA Astrophysics Data System (ADS)

Nanoscale pores can be used to analyze individual DNA or RNA molecules. For example, a prototype device based on the alpha-hemolysin protein permits serial examination of hundreds to thousands of molecules per minute. It is routinely used to identify individual polynucleotide homopolymers, and to read segments within single DNA or RNA block copolymers as they thread through a narrow, trans-membrane pore formed by the protein (1.5 nm limiting aperture). In recent reports, we have shown that this device can also be used to examine structural details of individual DNA or RNA hairpins at single base-pair precision. This is achieved by capturing each hairpin in a vestibule of the channel leading to the trans-membrane pore. Under a 120 mV applied voltage, ionic current through the channel is gated by the RNA or DNA hairpin as it is perched in the vestibule, resulting in a dynamic current pattern that is exquisitely sensitive to sequence identity. In this presentation, I will explain the ion channel device and then describe structural details of the hairpin molecules that can be resolved by the instrument. These include: i) Watson-Crick base-pair identity at the hairpin stem terminus; ii) duplex fraying caused by single base pair mismatches internal to the hairpin stem; and iii) A form (RNA) versus B form (DNA) helix conformation. I will then discuss alternative mechanisms that can account for the discrete gating patterns that underlie the analysis.

Akeson, Mark

2002-03-01

204

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases.  

PubMed

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed. PMID:10827184

Ma, W P; Kaiser, M W; Lyamicheva, N; Schaefer, J J; Allawi, H T; Takova, T; Neri, B P; Lyamichev, V I

2000-08-11

205

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

NASA Astrophysics Data System (ADS)

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

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

2005-08-01

206

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

PubMed Central

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5’ gene region or promoter hypermethylation, satellite or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, 6 of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P<0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor cause of satellite and global DNA hypomethylation.

Ehrlich, Melanie; Woods, Christian B.; Yu, Mimi C.; Dubeau, Louis; Yang, Fan; Campan, Mihaela; Weisenberger, Daniel J.; Long, Tiffany I.; Youn, Byungwoo; Fiala, Emerich S.; Laird, Peter W.

2005-01-01

207

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

PubMed Central

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

JIMENEZ-WENCES, HILDA; PERALTA-ZARAGOZA, OSCAR; FERNANDEZ-TILAPA, GLORIA

2014-01-01

208

Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.  

PubMed Central

A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently. Images

Thomas, P S

1980-01-01

209

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

PubMed

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

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

2014-06-01

210

Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule.  

PubMed

The interaction of DNA replication forks with both stationary and transcribing RNA polymerase molecules has been examined in vitro, using the multienzyme T4 bacteriophage DNA replication system and purified E. coli RNA polymerase. We have found that a single stationary RNA polymerase molecule can block the movement of the T4 replication fork when bound to a promoter on a double-stranded fd DNA template. When transcription is allowed (in the same direction as replication), the replication fork appears to follow the moving RNA polymerase molecule at the relatively slow rate of transcription. The barriers to fork movement formed by E. coli RNA polymerase are eliminated by the addition of small amounts of a purified T4-encoded DNA helicase, the product of the dda gene. We find that replication complexes containing the dda protein cause stationary RNA polymerase molecules to dissociate from the DNA. PMID:6136341

Bedinger, P; Hochstrasser, M; Jongeneel, C V; Alberts, B M

1983-08-01

211

Two regions in human DNA polymerase beta mRNA suppress translation in Escherichia coli.  

PubMed Central

Although human DNA polymerase beta (DNA pol beta) shows 96% identity with rat DNA pol beta at the amino acid level, it is weakly expressed in Escherichia (E.) coli relative to the rat enzyme. The mechanism of this suppression was investigated. Pulse-chase protein labeling and steady state mRNA analysis showed that mature human DNA pol beta protein is relatively stable in E. coli and the levels of human and rat DNA pol beta mRNA were comparable indicating that the human DNA pol beta expression is suppressed at the translational level. By systematic expression analysis of a number of chimeric genes composed of human and rat cDNAs, two strong translational suppression regions were mapped in the human DNA pol beta mRNA; one was named TSR-1, corresponding to CGG encoding arginine (arg) at position 4 and the other, termed TSR-2, is located between codons 153 and 199. Since substitution of the rat Arg-4 codon with synonymous codons showed strong effects upon the expression level, we propose that the arg codon at the N-terminal coding region plays a role in modulating expression. Images

Date, T; Tanihara, K; Yamamoto, S; Nomura, N; Matsukage, A

1992-01-01

212

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

PubMed Central

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

2013-01-01

213

The chemical synthesis of DNA/RNA: our gift to science.  

PubMed

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

Caruthers, Marvin H

2013-01-11

214

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

PubMed

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

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

2014-03-18

215

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

PubMed Central

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

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

2012-01-01

216

Configurational diffusion of coal macromolecules  

SciTech Connect

During this quarter, progress has been made in two areas; (1) review of the theories concerning hindered diffusion of large molecules and (2) measurement of effective diffusion coefficients for tetraphenylporphine (TPP). Experimental data of TPP diffusion experiments showed definite hindrance effects. However, direct comparison of experimental data to the theoretical prediction was not made at this time because of material balance errors in some experimental sets. This study will be continued next quarter and hindered diffusion experiments with coal macromolecules will also conducted. 7 refs., 3 figs.

Guin, J.A.; Curtis, C.W.; Tarrer, A.R.

1990-01-01

217

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

PubMed Central

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

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

2013-01-01

218

Genome-Wide Identification of Human RNA Editing Sites by Parallel DNA Capturing and Sequencing  

Microsoft Academic Search

Adenosine-to-inosine (A-to-I) RNA editing leads to transcriptome diversity and is important for normal brain function. To date, only a handful of functional sites have been identified in mammals. We developed an unbiased assay to screen more than 36,000 computationally predicted nonrepetitive A-to-I sites using massively parallel target capture and DNA sequencing. A comprehensive set of several hundred human RNA editing

Jin Billy Li; Erez Y. Levanon; Jung-Ki Yoon; John Aach; Bin Xie; Emily LeProust; Kun Zhang; Yuan Gao; George M. Church

2009-01-01

219

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

PubMed

The compact genome of the unicellular eukaryote Paramecium tetraurelia contains noncoding DNA (ncDNA) distributed into >39,000 intergenic sequences and >90,000 introns of 390 base pairs (bp) and 25 bp on average, respectively. Here we analyzed the molecular features of the ncRNA genes, introns, and intergenic sequences of this genome. We mainly used computational programs and comparative genomics possible because the P. tetraurelia genome had formed throughout whole-genome duplications (WGDs). We characterized 417 5S rRNA, snRNA, snoRNA, SRP RNA, and tRNA putative genes, 415 of which map within intergenic sequences, and two, within introns. The evolution of these ncRNA genes appears to have mainly involved purifying selection and gene deletion. We then compared the introns that interrupt the protein-coding gene duplicates arisen from the recent WGD and identified a population of a few thousands of introns having evolved under most stringent constraints (>95% of identity). We also showed that low nucleotide substitution levels characterize the 50 and 80-115 base pairs flanking, respectively, the stop and start codons of the protein-coding genes. Lower substitution levels mark the base pairs flanking the highly transcribed genes, or the start codons of the genes of the sets with a high number of WGD-related sequences. Finally, adjacent to protein-coding genes, we characterized 32 DNA motifs able to encode stable and evolutionary conserved RNA secondary structures and defining putative expression controlling elements. Fourteen DNA motifs with similar properties map distant from protein-coding genes and may encode regulatory ncRNAs. PMID:19218550

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

2009-04-01

220

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

PubMed Central

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

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

2009-01-01

221

Early Growth of Wheat Embryonic Axes and the Synthesis of RNA and DNA 1  

PubMed Central

The requirement for the synthesis of RNA and DNA in early germination of wheat (Triticum aestivum var Newana) embryonic axes has been studied by incubating embryos in the presence of appropriate inhibitors and monitoring both embryo growth and the rates of specific metabolic processes. Experiments with 5-fluorouridine showed that both rRNA and DNA synthesis could be curtailed by 60 to 70% without affecting embryo growth to 24 hours. Similarly, the presence of mitomycin C and methotrexate inhibited DNA synthesis 70%, with only a small effect on growth. Experiments with a range of concentrations of cordycepin and ?-amanitin indicated that mRNA synthesis could be curtailed by 30 to 40% within the first 8 hours of germination with only a small effect on embryo growth. Thus, at least the initial phases of seed embryo germination are not closely linked to the synthesis of mRNA, rRNA, or DNA. Maximal sensitivity of embryo growth was obtained with cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, supporting the idea that protein synthesis is the macromolecular process most closely linked to early germination.

Datta, Ketaki; Marsh, Loren; Marcus, Abraham

1983-01-01

222

Catalytic Activity of a Binary Informational Macromolecule  

NASA Technical Reports Server (NTRS)

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

Reader, John S.; Joyce, Gerald F.

2003-01-01

223

Direct Evidence that RNA Inhibits APOBEC3G ssDNA Cytidine Deaminase Activity  

PubMed Central

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

McDougall, William M.; Smith, Harold C.

2011-01-01

224

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

PubMed

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

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

2014-07-01

225

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

Microsoft Academic Search

The generation of reactive oxygen species is thought to cause extensive oxidative damage to various biomolecules such as DNA,\\u000a RNA, and protein. In this study, the preventive, suppressive, and protective effects of in vitro supplementation with electrolyzed-reduced\\u000a water on H2O2-induced DNA damage in human lymphocytes were examined using a comet assay. Pretreatment, cotreatment, and posttreatment with\\u000a electrolyzed-reduced water enhanced human

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

2006-01-01

226

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

Microsoft Academic Search

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

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

1990-01-01

227

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

Microsoft Academic Search

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

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

1994-01-01

228

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.  

PubMed Central

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.

Kramer, M G; Khan, S A; Espinosa, M

1997-01-01

229

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

PubMed Central

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

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

2010-01-01

230

Multifunctional Iron Oxide Nanoparticles for Diagnostics, Therapy and Macromolecule Delivery  

PubMed Central

In recent years, multifunctional nanoparticles (NPs) consisting of either metal (e.g. Au), or magnetic NP (e.g. iron oxide) with other fluorescent components such as quantum dots (QDs) or organic dyes have been emerging as versatile candidate systems for cancer diagnosis, therapy, and macromolecule delivery such as micro ribonucleic acid (microRNA). This review intends to highlight the recent advances in the synthesis and application of multifunctional NPs (mainly iron oxide) in theranostics, an area used to combine therapeutics and diagnostics. The recent applications of NPs in miRNA delivery are also reviewed.

Yen, Swee Kuan; Padmanabhan, Parasuraman; Selvan, Subramanian Tamil

2013-01-01

231

Multifunctional iron oxide nanoparticles for diagnostics, therapy and macromolecule delivery.  

PubMed

In recent years, multifunctional nanoparticles (NPs) consisting of either metal (e.g. Au), or magnetic NP (e.g. iron oxide) with other fluorescent components such as quantum dots (QDs) or organic dyes have been emerging as versatile candidate systems for cancer diagnosis, therapy, and macromolecule delivery such as micro ribonucleic acid (microRNA). This review intends to highlight the recent advances in the synthesis and application of multifunctional NPs (mainly iron oxide) in theranostics, an area used to combine therapeutics and diagnostics. The recent applications of NPs in miRNA delivery are also reviewed. PMID:24396508

Yen, Swee Kuan; Padmanabhan, Parasuraman; Selvan, Subramanian Tamil

2013-01-01

232

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

SciTech Connect

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

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

2012-10-23

233

Photoregulation of biologically active macromolecules.  

PubMed

A broad view is given of photoregulated processes as they occur in algae, fungi, halophilic bacteria, higher plants, invertebrates, and higher animals. Emphasis is on the following: the organs, tissues, and organelles that participate; the nature of the photoreceptor pigments; the light-induced structural changes that occur in the photopigments; and the way in which the photochemical events are believed to be translated into the physiological response. An attempt is made to show that there exist common biochemical attributes in all systems. In particular, they depend upon the ability of a low-molecular-weight to regulate a biologically active macromolecule, which may or may not be incorporated into a membrane. This is a common type of biochemical regulation and is, for example, the basis of allosterism. The additional refinement in photosensitive systems is the ability of light to alter the stereochemistry of the low-molecular-weight effector molecule and thus to modify its effect on the macromolecule. Model photosensitive systems are examined that incorporate control mechanisms that function in natural systems. For example, there are systems in which enzymes, normally insensitive to light, are made subject to photoregulation. In others, membrane permeability is rendered photoresponsive. A comparison of the model systems was processes found in nature permits the formulation of an hypothesis to explain how naturally occurring photoresponsive systems might have evolved. PMID:822780

Erlanger, B F

1976-01-01

234

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.  

PubMed

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe. PMID:17720718

Mukai, Hiroyuki; Uemori, Takashi; Takeda, Osamu; Kobayashi, Eiji; Yamamoto, Junko; Nishiwaki, Kazue; Enoki, Tatsuji; Sagawa, Hiroaki; Asada, Kiyozo; Kato, Ikunoshin

2007-08-01

235

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

PubMed Central

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

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

1998-01-01

236

Charity begins at home: non-coding RNA contributes to DNA repair  

PubMed Central

During the past decade, evolutionarily conserved non-coding (nc) RNAs, specifically microRNAs (miRNA), have been characterized as regulators of almost every cellular process and signalling pathway. There is now emerging evidence that this new class of regulators also impinges on the DNA damage response (DDR). Both miRNAs and other small ncRNAs are induced at DNA breaks and mediate the repair process. These intriguing observations raise the possibility that crosstalk between ncRNAs and the DDR might provide a means of efficient and accurate DNA repair and facilitate the maintenance of genomic stability.

Chowdhury, Dipanjan; Choi, Young Eun; Brault, Marie Eve

2014-01-01

237

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

PubMed

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

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

2014-08-01

238

Age-Related Changes in the DNA and RNA Content of the Brain in Spontaneously Hypertensive Rats  

Microsoft Academic Search

To elucidate the effects of aging accompanied with hypertension on brain nucleic acid, we measured both the DNA and RNA contents of six specific brain regions in adult (5–6 months old) and aged (18–22 months old) female spontaneously hypertensive rats (SHRs). Although no statistical difference was observed in the RNA content, the DNA content did tend to increase in the

Hiroshi Nakane; Tatsuo Nakahara; Hiroshi Yao; Hiroaki Ooboshi; Setsuro Ibayashi; Hideyuki Uchimura; Masatoshi Fujishima

1997-01-01

239

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

PubMed Central

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

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

2010-01-01

240

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

Microsoft Academic Search

This study was carried out to assess the effects of 14 trace elements, 12 herbicides, and two fungicides on dephosphorylation of yeast ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) added to soils (Xerollic Calciorthids and Typic Haploxeralfs). The cumulative amount of ortho phosphate (Pi) released from nucleic acids increased linearly with time of incubation (up to 72 h), decreased with

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

1986-01-01

241

Inhibition by carbaryl of DNA, RNA and protein synthesis in cultured rat lung cells  

Microsoft Academic Search

Summary The carbamate pesticide carbaryl rapidly inhibited DNA, RNA and protein synthesis in L-2 cells from rat lung. the inhibiton was partly reversible and was not accompanied by inhibiton of transport of tritiated precursors into intracellular pools or destruction of the integrity of the cell membrane.

J. M. Lockard; B. P. Schuette; P. S. Sabharwal

1982-01-01

242

Pyrrole-imidazole polyamides distinguish between double-helical DNA and RNA.  

PubMed

Groove specificity: pyrrole-imidazole polyamides are well-known for their specific interactions with the minor groove of DNA. However, polyamides do not show similar binding to duplex RNA, and a structural rationale for the molecular-level discrimination of nucleic acid duplexes by minor-groove-binding ligands is presented. PMID:22987334

Chenoweth, David M; Meier, Jordan L; Dervan, Peter B

2013-01-01

243

Association between Urinary Excretion of Cortisol and Markers of Oxidatively Damaged DNA and RNA in Humans  

Microsoft Academic Search

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the

Anders Joergensen; Kasper Broedbaek; Allan Weimann; Richard D. Semba; Luigi Ferrucci; Martin B. Joergensen; Henrik E. Poulsen

2011-01-01

244

Association between Urinary Excretion of Cortisol and Markers of Oxidatively Damaged DNA and RNA in Humans  

PubMed Central

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the relationship between 24 h excretion of urinary cortisol and markers of oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2?-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, in a sample of 220 elderly men and women (age 65 – 83 years). We found a robust association between the excretion of cortisol and the oxidation markers (R2?=?0.15, P<0.001 for both markers). Individuals in the highest quartile of cortisol excretion had a 57% and 61% higher median excretion of the DNA and RNA oxidation marker, respectively, than individuals in the lowest quartile. The finding adds support to the hypothesis that cortisol-induced damage to DNA/RNA is an explanatory factor in the complex relation between stress, aging and disease.

Joergensen, Anders; Broedbaek, Kasper; Weimann, Allan; Semba, Richard D.; Ferrucci, Luigi; Joergensen, Martin B.; Poulsen, Henrik E.

2011-01-01

245

Effect of granaticin on the activity of DNA-directed RNA polymerase from Streptomyces granaticolor.  

PubMed

DNA-directed RNA polymerase was isolated from wild-type and mutant (asporogenic and granaticin overproducing) Streptomyces granaticolor. The effect of granaticin on the enzyme activity was compared with that of standard transcription inhibitors, rifampicin and rifamycin SV and shown to be zero. PMID:2635132

Smardová, J; Spízek, J; Stastná, J; Klégr, M

1989-01-01

246

Differential DNA and RNA sequence discrimination by PNA having charged side chains.  

PubMed

PNA sequences modified with charged side chains were evaluated for base-pairing sequence selectivity under physiological conditions. PNA having negatively charged aspartic acid side chains shows higher selectivity with RNA, while PNA having positively charged lysine side chains shows higher selectivity with DNA. These observations provide insight into the binding selectivity of modified PNA in antisense and antigene applications. PMID:24731279

De Costa, N Tilani S; Heemstra, Jennifer M

2014-05-15

247

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

PubMed Central

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

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

2013-01-01

248

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

Microsoft Academic Search

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

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

1996-01-01

249

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

PubMed Central

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

Arima, Hidetoshi; Motoyama, Keiichi

2009-01-01

250

Aberrant DNA methylation of microRNA genes in human breast cancer - a critical appraisal.  

PubMed

Aberrant DNA methylation of regulatory sequences is a well-documented mechanism of functional deletion of genes with anti-tumourigenic properties including microRNAs. This review discusses the publications describing aberrant methylation of microRNA genes in human breast cancer cells. Among the anti-tumourigenic properties of epigenetically inactivated microRNA genes, the inhibition of proliferation and of epithelial-to-mesenchymal transition (EMT) are the best studied. Several studies are conceptually very interesting and present a comprehensive functional characterization of anti-tumorigenic microRNAs. The link between microRNA expression and gene methylation is not addressed directly by all studies and a number of studies are limited in their strength by not including primary breast cancer specimens or by analysing very small sets of primary human specimens. The publications cover a wide range of DNA methylation detection techniques, often making direct comparison of results challenging. Despite the identification and thorough characterization of many interesting candidates and functionally important microRNA genes affected by DNA methylation, the translation of microRNA gene methylation as a new biomarker into the daily routine practice has not yet worked out. PMID:24509818

Lehmann, Ulrich

2014-06-01

251

Large accumulation of mRNA and DNA point modifications in a plant senescent tissue.  

PubMed

Although nucleic acids are the paradigm of genetic information conservation, they are inherently unstable molecules that suffer intrinsic and environmental damage. Oxidative stress has been related to senescence and aging and, recently, it has been shown that mutations accumulate at high frequency in mitochondrial DNA with age. We investigated RNA and DNA modifications in cork, a senescent plant tissue under high endogenous oxidative stress conditions. When compared to normally growing young tissue, cork revealed an unexpected high frequency of point modifications in both cDNA (Pn = 1/1784) and nuclear DNA (Pn = 1/1520). Cork should be viewed as a mosaic of genetically heterogeneous cells. This has biological implications: it supports somatic mutation models for aging and challenges 'single cDNA clone' as descriptor for the molecular genetics of senescent tissues. PMID:10781796

Pla, M; Jofré, A; Martell, M; Molinas, M; Gómez, J

2000-04-21

252

Delivery of Drugs and Macromolecules to Mitochondria  

Microsoft Academic Search

Mitochondria is where the bulk of the cell's ATP is produced. Mutations occur to genes coding for members of the complexes involved in energy production. Some are a result of damages to nuclear coded genes and others to mitochondrial coded genes. This review describes approaches to bring small molecules, proteins and RNA\\/DNA into mitochondria. The purpose is to repair damaged

Abhijit Mukhopadhyay; Henry Weiner

2008-01-01

253

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

Microsoft Academic Search

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

Panarat Tomanee; James T. Hsu

2006-01-01

254

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

SciTech Connect

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

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

2012-12-01

255

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

PubMed Central

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

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

2013-01-01

256

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

PubMed

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

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

2013-06-11

257

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

PubMed

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

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

2014-08-01

258

Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA.  

PubMed

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA. PMID:23000270

Calarco, Joseph P; Borges, Filipe; Donoghue, Mark T A; Van Ex, Frédéric; Jullien, Pauline E; Lopes, Telma; Gardner, Rui; Berger, Frédéric; Feijó, José A; Becker, Jörg D; Martienssen, Robert A

2012-09-28

259

Molecular logic gates on DNA origami nanostructures for microRNA diagnostics.  

PubMed

Molecular computing holds great promise for diagnosis and treatment of diseases at the molecular level; nevertheless, designing molecular logic gates to operate programmably and autonomously for molecular diagnostics still remains challenging. We designed logic gates on DNA Origami for microRNA analysis. As a demonstration, two indicators of heart failure, microRNA-21 and microRNA-195, were selected as the logic inputs. The logic gates contain two main modules: computation module and output module, performing in a single DNA Origami nanostructure. The computation module recognizes disease indicators, while the output module display different nanoscale symbols, "+" (positive) or "-" (negative), depending on the computing results. We demonstrated that the molecular logic gates worked well with single and two input combinations. PMID:24447268

Wang, Dongfang; Fu, Yanming; Yan, Juan; Zhao, Bin; Dai, Bin; Chao, Jie; Liu, Huajie; He, Dannong; Zhang, Yi; Fan, Chunhai; Song, Shiping

2014-02-18

260

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

PubMed Central

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

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

2012-01-01

261

Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants  

PubMed Central

Background DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known. Results By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin. Conclusions We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

2014-01-01

262

MACROMOLECULES FACILITATE THE TRANSPORT OF TRACE ORGANICS  

EPA Science Inventory

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

263

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

PubMed

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

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

1977-09-01

264

A computer program package for storing and retrieving DNA/RNA and protein sequence data.  

PubMed Central

A computer program package has been made available which contains five compound programs written in FORTRAN 10 (for DECsystem-10). Program DATBAS is for storing and improving DNA sequence data, especially those obtained by the sequencing method using M13 phages. Programs NUCDAT and PROTEN are for analyzing DNA/RNA and protein sequence data, respectively. They contain various options to help users in analyzing DNA/RNA and protein sequence data. With program NUCDAT, it is also possible to get access to the EMBO Nucleotide Data Library. This can be achieved by running program COPY to create two data files from the library. Program LITRAT enables users to prepare a scientific literature file convenient for writing scientific articles, and program STRAIN for storing information concerning bacterial and/or plasmid strains.

Isono, K

1984-01-01

265

New molecular diagnosis and screening methods for colorectal cancer using fecal protein, DNA and RNA.  

PubMed

Several screening methods for reducing the mortality rate of colorectal cancer (CRC) have been reported in recent decades. Fecal occult blood tests (FOBTs) are widely used for CRC screening and immunochemical FOBTs perform better than guaiac FOBTs; however, the sensitivity and specificity of immunochemical FOBTs remain unsatisfactory. To resolve this problem, novel fecal molecular methods based on fecal protein, DNA and RNA analyses have been developed. Regarding fecal proteins, several marker proteins indicating intestinal bleeding and cancer cell-specific proteins have been investigated. Regarding fecal DNA, numerous gene mutation and gene methylation analyses have been reported. Consequently, fecal DNA analysis was recommended as a CRC screening method in 2008. In addition, gene expression analyses of CRC-specific genes and miRNAs in fecal RNA have been investigated over the last decade. This review article summarizes molecular methods using fecal samples for CRC screening, focusing on reports within the last 5 years. PMID:24308334

Koga, Yoshikatsu; Yamazaki, Nobuyoshi; Matsumura, Yasuhiro

2014-01-01

266

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

PubMed

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

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

2009-01-01

267

Circular dichroism of biological macromolecules.  

PubMed

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

Beychok, S

1966-12-01

268

Correlation functions of flexible macromolecules  

NASA Astrophysics Data System (ADS)

We present an ab initio calculation of conformational entropy, radii of gyration, X-ray and neutron scattering form-factors, correlation functions, and other observables describing proteins, polypeptides, nucleic acids, related macromolecules and artificial polymers [1]. The analytic form of the method minimizes computational costs and reveals relations between observables. We apply these methods to study thermodynamics of protein unfolding. The presented results agree with the results from experiment and simulation [2]. [1] O.K.Vorov, A.Y.Istomin, D.R.Livesay, D.J.Jacobs, subm. to Phys.Rev.Lett., 2007. [2] O.K.Vorov, D.R.Livesay, D.J.Jacobs, to be subm. to Proc. Natl. Acad. Sci., 2007, in preparation.

Jacobs, Donald; Livesay, Dennis; Vorov, Oleg

2008-03-01

269

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor  

PubMed Central

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Kanno, Tatsuo; Bucher, Etienne; Daxinger, Lucia; Huettel, Bruno; Kreil, David P; Breinig, Frank; Lind, Marc; Schmitt, Manfred J; Simon, Stacey A; Gurazada, Sai Guna Ranjan; Meyers, Blake C; Lorkovic, Zdravko J; Matzke, Antonius J M; Matzke, Marjori

2010-01-01

270

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

PubMed Central

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

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

2012-01-01

271

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA.  

PubMed

DNA damage, stalled replication forks, errors in mRNA splicing, and availability of nutrients activate specific phosphatidylinositiol-3 kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2, or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double-strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA. PMID:21701263

Gewandter, Jennifer S; Bambara, Robert A; O'Reilly, Michael A

2011-08-01

272

The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA  

PubMed Central

DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.

Gewandter, Jennifer S; Bambara, Robert A

2011-01-01

273

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

PubMed Central

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

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

2012-01-01

274

Fluorescence-, isotope- or biotin-labeling of the 5 '-end of single-stranded DNA/RNA using T4 RNA ligase.  

PubMed Central

A rapid 5'-labeling method of single-stranded DNA/RNA was developed, which is based on the utilization of an adenylated intermediate in the reaction of T4 RNA ligase. This method is commonly useful for fluorescence-, isotope- or biotin-labeling of the 5'-ends of both oligo- and polynucleotides.

Kinoshita, Y; Nishigaki, K; Husimi, Y

1997-01-01

275

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 ...

276

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 ...

277

Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors.  

PubMed Central

The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity. These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase. This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J. Laffan and W. Firshein, J. Bacteriol. 169:2819-2827, 1987). Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation. Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives. Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase. Some regulatory mechanisms of the kinase system may also be retained by the complex. Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex. Images

Laffan, J J; Skolnik, I L; Hadley, D A; Bouyea, M; Firshein, W

1990-01-01

278

Quantification of Pregenomic RNA and Covalently Closed Circular DNA in Hepatitis B Virus-Related Hepatocellular Carcinoma  

PubMed Central

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

Bai, Fugui; Yano, Yoshihiko; Fukumoto, Takumi; Takebe, Atsushi; Tanaka, Motofumi; Anggorowati, Nungki; Widasari, Dewiyani Indah; Saito, Masaya; Hirano, Hirotaka; Seo, Yasushi; Azuma, Takeshi; Ku, Yonson

2013-01-01

279

Roles for Pbp1 and Caloric Restriction in Genome and Lifespan Maintenance via Suppression of RNA-DNA Hybrids.  

PubMed

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

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

280

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

PubMed Central

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.

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

281

Efficient integration of an intron RNA into double-stranded DNA by reverse splicing.  

PubMed

Some group II introns are mobile elements as well as catalytic RNAs. Introns aI1 and aI2 found in the gene COX1 in yeast mitochondria encode reverse transcriptases which promote site-specific insertion of the intron into intronless alleles ('homing'). For aI2 this predominantly occurs by reverse transcription of unspliced precursor RNA at a break in double-strand DNA made by an endonuclease encoded by the intron. The aI2 endonuclease involves both the excised intron RNA, which cleaves the DNA's sense strand by partial reverse splicing; and the intron-encoded reverse transcriptase which cleaves the anti-sense strand. Here we show that aI1 encodes an analogous endonuclease specific for a different target site compatible with the different exon-binding sequences of the intron RNA. Over half of aI1 undergoes complete reverse splicing in vitro, thus integrating linear intron RNA directly into the DNA. This unprecedented reaction has implications for both intron mobility and evolution, and potential genetic engineering applications. PMID:8692273

Yang, J; Zimmerly, S; Perlman, P S; Lambowitz, A M

1996-05-23

282

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

PubMed Central

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

Gilmour, D S; Lis, J T

1984-01-01

283

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.  

PubMed

A lambda gt11 library constructed from poly(A+) 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'-untranslated region. A subsequent screen using a 5' 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. PMID:1702999

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

1990-10-16

284

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.  

PubMed

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

2007-09-01

285

Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1.  

PubMed

DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a Pol-IV-interacting protein. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin-binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals, a further understanding of this early targeting step may aid our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy. PMID:23636332

Law, Julie A; Du, Jiamu; Hale, Christopher J; Feng, Suhua; Krajewski, Krzysztof; Palanca, Ana Marie S; Strahl, Brian D; Patel, Dinshaw J; Jacobsen, Steven E

2013-06-20

286

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

PubMed

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

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

2014-01-01

287

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

PubMed Central

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

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

1994-01-01

288

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

PubMed Central

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

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

2012-01-01

289

Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes  

Microsoft Academic Search

Double-stranded RNA (dsRNA) induces sequence- specific mRNA degradation in most eukaryotic organisms via a conserved pathway known as RNA interference (RNAi). Post-transcriptional gene silen- cing by RNAi is also connected with transcriptional silencing of cognate sequences. In plants, this tran- scriptional silencing is associated with sequence- specific DNA methylation. To address whether this mechanism operates in mammalian cells, we used

Petr Svoboda; Paula Stein; Witold Filipowicz; Richard M. Schultz

2004-01-01

290

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

PubMed Central

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

2013-01-01

291

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

PubMed Central

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

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

2014-01-01

292

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...

293

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

SciTech Connect

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

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

2009-03-10

294

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

SciTech Connect

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

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

2009-11-20

295

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles.  

PubMed

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ?-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy. PMID:21983283

Vader, P; Crielaard, B J; van Dommelen, S M; van der Meel, R; Storm, G; Schiffelers, R M

2012-06-10

296

Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation  

PubMed Central

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with ?1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with ?5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

Revyakin, Andrey; Ebright, Richard H.; Strick, Terence R.

2004-01-01

297

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

PubMed Central

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

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

2013-01-01

298

Rigidity of Glasses and Macromolecules  

NASA Astrophysics Data System (ADS)

The simple yet powerful ideas of percolation theory have found their way into many different areas of research. In this talk we show how RIGIDITY PERCOLATION can be studied at a similar level of sophistication, using a powerful new program THE PEBBLE GAME (D. J. Jacobs and M. F. Thorpe, Phys. Rev. E) 53, 3682 (1996). that uses an integer algorithm. This program can analyse the rigidity of two and three dimensional networks containing more than one million bars and joints. We find the total number of floppy modes, and find the critical behavior as the network goes from floppy to rigid as more bars are added. We discuss the relevance of this work to network glasses, and how it relates to experiments that involve the mechanical properties like hardness and elasticity of covalent glassy networks like Ge_xAs_ySe_1-x-y and dicuss recent experiments that suggest that the rigidity transition may be first order (Xingwei Feng, W. J.Bresser and P. Boolchand, Phys. Rev. Lett 78), 4422 (1997).. This approach is also useful in macromolecules and proteins, where detailed information about the rigid domain structure can be obtained.

Thorpe, M. F.

1998-03-01

299

DNA and RNA analyses in detection of genetic predisposition to cancer  

PubMed Central

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

2012-01-01

300

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

PubMed Central

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

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

2012-01-01

301

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

NASA Astrophysics Data System (ADS)

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.

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

302

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

Microsoft Academic Search

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

Lakshminarayan M Iyer; Eugene V Koonin; L Aravind

2003-01-01

303

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

SciTech Connect

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

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

1985-07-01

304

Synergistic Activation of Innate Immunity by Double-Stranded RNA and CpG DNA Promotes Enhanced Antitumor Activity  

Microsoft Academic Search

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Com- bined treatments demonstrated synergy in nitric oxide,

Mark M. Whitmore; Michael J. DeVeer; Andrea Edling; Rhonda K. Oates; Brenna Simons; Daniel Lindner; Bryan R. G. Williams

2004-01-01

305

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

SciTech Connect

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

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

1986-01-01

306

Maize NADP-malate dehydrogenase: cDNA cloning, sequence, and mRNA characterization  

Microsoft Academic Search

We report here the isolation, characterization and nucleotide sequence of clones encoding the maize chloroplastic NADP-malate dehydrogenase (NADP-MDH) which functions in the C4 cycle of photosynthesis. A nearly full-length NADP-MDH cDNA clone was isolated using antibodies against the purified protein. This clone hybridizes to a 1600 base mRNA that is eight times more abundant in light-grown maize leaves than in

Mary C. Metzler; Beverly A. Rothermel; Timothy Nelson

1989-01-01

307

A simple and direct electrochemical detection of interferon-? using its RNA and DNA aptamers  

Microsoft Academic Search

Tuberculosis is the most frequent cause of infection-related death worldwide. We constructed a simple and direct electrochemical sensor to detect interferon (IFN)-?, a selective marker for tuberculosis pleurisy, using its RNA and DNA aptamers. IFN-? was detected by its 5?-thiol-modified aptamer probe immobilized on the gold electrode. Interaction between IFN-? and the aptamer was recorded using electrochemical impedance spectroscopy and

Kyoungin Min; Minseon Cho; Se-Young Han; Yoon-Bo Shim; Jakang Ku; Changill Ban

2008-01-01

308

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

PubMed

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

Chícharo, Maria A; Chícharo, Luis

2008-08-01

309

Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age  

PubMed Central

Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations.

Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M.; Stewart, James B.; Bj?ras, Magnar; Eide, Lars

2014-01-01

310

IMB Jena: Image Library of Biological Macromolecules  

NSDL National Science Digital Library

The Institute for Molecular Biotechnology (Jena, Germany) provides this repository of molecular structures, featuring selected VRML (as well as two dimensional) images of "building blocks," and "macromolecular structures." Included among the images are RNA structures from the protein database at the Brookhaven National Laboratory, as well as over 250 DNA and protein structures. Sequence and descriptive information about the images is available. Non-VRML images are searchable.

Suhnel, Jurgen.

311

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

PubMed Central

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

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

2013-01-01

312

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

PubMed Central

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

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

2010-01-01

313

DNA Damage-Dependent Transcriptional Arrest and Termination of RNA Polymerase II Elongation Complexes in DNA Template Containing HIV1 Promoter  

Microsoft Academic Search

We have developed a new biochemical method to isolate a homogenous population of RNA polymerase II (RNA pol II) elongation complexes arrested at a DNA damage site. The method involves triple-helix formation at a predetermined site in DNA template with a third strand labeled with psoralen at its 5'-end and a biotin at the 3'-end. After triplex formation and near-ultraviolet

Zhuying Wang; Tariq M. Rana

1997-01-01

314

Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.  

PubMed

The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding. PMID:11691931

Chennathukuzhi, V M; Kurihara, Y; Bray, J D; Yang, J; Hecht, N B

2001-11-01

315

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

NASA Astrophysics Data System (ADS)

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

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

316

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

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

317

RNA-directed DNA methylation: an epigenetic pathway of increasing complexity.  

PubMed

RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. RdDM requires a specialized transcriptional machinery that comprises two plant-specific RNA polymerases - Pol IV and Pol V - and a growing number of accessory proteins, the functions of which in the RdDM mechanism are only partially understood. Recent work has revealed variations in the canonical RdDM pathway and identified factors that recruit Pol IV and Pol V to specific target sequences. RdDM, which transcriptionally represses a subset of transposons and genes, is implicated in pathogen defence, stress responses and reproduction, as well as in interallelic and intercellular communication. PMID:24805120

Matzke, Marjori A; Mosher, Rebecca A

2014-06-01

318

mRNA and DNA Detection of Human Papillomaviruses in Women of All Ages Attending Two Colposcopy Clinics  

PubMed Central

Objective HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. Methods 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. Results HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests’ positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. Conclusions Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results.

Spathis, Aris; Kottaridi, Christine; Chranioti, Aikaterini; Meristoudis, Christos; Chrelias, Charalambos; Panayiotides, Ioannis G.; Paraskevaidis, Evangelos; Karakitsos, Petros

2012-01-01

319

PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.  

PubMed

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary. PMID:15322425

Byers, Richard; Roebuck, Jamie; Sakhinia, Ebrahim; Hoyland, Judith

2004-09-01

320

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

PubMed

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

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

2012-01-01

321

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

PubMed Central

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

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

2005-01-01

322

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

PubMed Central

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

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

2013-01-01

323

Macromolecule-Assisted de novo Protein Folding  

PubMed Central

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

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

2012-01-01

324

CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing.  

PubMed

Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks. PMID:21964334

Shukla, Sanjeev; Kavak, Ersen; Gregory, Melissa; Imashimizu, Masahiko; Shutinoski, Bojan; Kashlev, Mikhail; Oberdoerffer, Philipp; Sandberg, Rickard; Oberdoerffer, Shalini

2011-11-01

325

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases.  

PubMed

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene. PMID:8837454

Dowjat, W K; Kharatishvili, M; Costa, M

1996-10-01

326

Inhibition of DNA and RNA Polymerase Reactions by Trypanocidal Drugs. Effect of Aromatic Diamidine and Phenanthridinium Compounds.  

National Technical Information Service (NTIS)

Several trypanocidal drugs were tested for possible inhibitory activity towards DNA (pol. I) and RNA polymerase. These chemotherapeutically active agents are throught to inhibit nucleic acid syntheses in African bloodstream trypanosomes. In the isolated D...

A. C. Zahalsky D. G. Brown N. F. Nelson

1979-01-01

327

RNA.  

ERIC Educational Resources Information Center

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

Darnell, James E., Jr.

1985-01-01

328

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

PubMed Central

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

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

2012-01-01

329

Interplay between Active Chromatin Marks and RNA-Directed DNA Methylation in Arabidopsis thaliana  

PubMed Central

DNA methylation is an epigenetic mark that is associated with transcriptional repression of transposable elements and protein-coding genes. Conversely, transcriptionally active regulatory regions are strongly correlated with histone 3 lysine 4 di- and trimethylation (H3K4m2/m3). We previously showed that Arabidopsis thaliana plants with mutations in the H3K4m2/m3 demethylase JUMONJI 14 (JMJ14) exhibit a mild reduction in RNA-directed DNA methylation (RdDM) that is associated with an increase in H3K4m2/m3 levels. To determine whether this incomplete RdDM reduction was the result of redundancy with other demethylases, we examined the genetic interaction of JMJ14 with another class of H3K4 demethylases: LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 1 and LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 2 (LDL1 and LDL2). Genome-wide DNA methylation analyses reveal that both families cooperate to maintain RdDM patterns. ChIP-seq experiments show that regions that exhibit an observable DNA methylation decrease are co-incidental with increases in H3K4m2/m3. Interestingly, the impact on DNA methylation was stronger at DNA-methylated regions adjacent to H3K4m2/m3-marked protein-coding genes, suggesting that the activity of H3K4 demethylases may be particularly crucial to prevent spreading of active epigenetic marks. Finally, RNA sequencing analyses indicate that at RdDM targets, the increase of H3K4m2/m3 is not generally associated with transcriptional de-repression. This suggests that the histone mark itself—not transcription—impacts the extent of RdDM.

Liu, Ao; Feng, Suhua; Jacobsen, Steven E.

2013-01-01

330

SELEX_DB: an activated database on selected randomized DNA\\/RNA sequences addressed to genomic sequence annotation  

Microsoft Academic Search

SELEX_DB is a novel curated database on selected randomized DNA\\/RNA sequences designed for accumulation of experimental data on functional site sequences obtained by using SELEX and SELEX-like technologies from the pools of random sequences. This database also contains the programs for DNA\\/RNA functional site recognition within arbitrary nucleotide sequences. The first release of SELEX_DB has been installed under SRS and

Julia V. Ponomarenko; Galina Orlova; Mikhail P. Ponomarenko; Sergey V. Lavryushev; Anatoly S. Frolov; Svetlana V. Zybova; Nikolay A. Kolchanov

2000-01-01

331

The RNA structure alignment ontology  

PubMed Central

Multiple sequence alignments are powerful tools for understanding the structures, functions, and evolutionary histories of linear biological macromolecules (DNA, RNA, and proteins), and for finding homologs in sequence databases. We address several ontological issues related to RNA sequence alignments that are informed by structure. Multiple sequence alignments are usually shown as two-dimensional (2D) matrices, with rows representing individual sequences, and columns identifying nucleotides from different sequences that correspond structurally, functionally, and/or evolutionarily. However, the requirement that sequences and structures correspond nucleotide-by-nucleotide is unrealistic and hinders representation of important biological relationships. High-throughput sequencing efforts are also rapidly making 2D alignments unmanageable because of vertical and horizontal expansion as more sequences are added. Solving the shortcomings of traditional RNA sequence alignments requires explicit annotation of the meaning of each relationship within the alignment. We introduce the notion of “correspondence,” which is an equivalence relation between RNA elements in sets of sequences as the basis of an RNA alignment ontology. The purpose of this ontology is twofold: first, to enable the development of new representations of RNA data and of software tools that resolve the expansion problems with current RNA sequence alignments, and second, to facilitate the integration of sequence data with secondary and three-dimensional structural information, as well as other experimental information, to create simultaneously more accurate and more exploitable RNA alignments.

Brown, James W.; Birmingham, Amanda; Griffiths, Paul E.; Jossinet, Fabrice; Kachouri-Lafond, Rym; Knight, Rob; Lang, B. Franz; Leontis, Neocles; Steger, Gerhard; Stombaugh, Jesse; Westhof, Eric

2009-01-01

332

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ 1  

PubMed Central

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH4)2SO4 precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000. Images

Jendrisak, Jerry

1981-01-01

333

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

PubMed Central

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

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

2013-01-01

334

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

PubMed

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

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

2014-08-01

335

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

PubMed

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

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

2014-04-01

336

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

PubMed

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

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

2010-09-01

337

DNA sequencing analysis of ITS and 28S rRNA of Poria cocos.  

PubMed

We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype. PMID:17666806

Atsumi, Toshiyuki; Kakiuchi, Nobuko; Mikage, Masayuki

2007-08-01

338

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

PubMed

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

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

2014-05-01

339

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking  

PubMed Central

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ?10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ?25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.

Xu, Jing; Jung, Jae Hoon; Marshall, Robert M.; McMahan, Uel J.

2013-01-01

340

RNA Pol II promotes transcription of centromeric satellite DNA in beetles.  

PubMed

Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera). This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80%) are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A) tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome. PMID:18270581

Pezer, Zeljka; Ugarkovi?, Durdica

2008-01-01

341

Dynamics and Stability of Individual Base Pairs in Two Homologous RNA-DNA Hybrids†  

PubMed Central

Nuclear magnetic resonance spectroscopy and proton exchange have been used to characterize two RNA-DNA hybrids from the tR2 intrinsic transcription terminator site of phage ?. The hybrids have the same base sequence: 5’-GGCGCAGGCC(T/U)(T/U)CC-3’/5’-GGAAGGCC(T/U)GCGCC-3’, but differ from each other by an interchange of DNA and RNA strands. The opening of single base pairs in the two hybrids is characterized by measuring the rates of exchange of imino protons with solvent protons as a function of the concentration of a proton acceptor (ammonia base) at 10 °C. The free energy change in the opening reaction provides a measure of the stability of the base pair, while the rates of opening and closing define the base-pair dynamics. The results demonstrate that, within the same base sequence context, dA-rU base pairs are less stable than dT-rA base pairs. The differences in stability are enhanced when two dA-rU base pairs are located next to each other in the hybrid structure. For the G-C base pairs, the rates of opening and closing, and the stability are affected by the base sequence context and by the nature of the sugar moiety attached to the guanine. The dominant feature of the base sequence is the proximity of the dA-rU base pair, which destabilizes the G-C base pair when the guanine is located on the DNA strand. Two G-C base pairs (namely, those in the 4th and 10th positions) exhibit large differences in their opening and closing rates between the two hybrids, while maintaining the same stability. These results provide the first demonstration that, for RNA-DNA hybrid structures with the same base sequence, the opening dynamics and the stability of individual base pairs is strongly influenced by the chemical nature of each strand.

Huang, Yuegao; Chen, Congju; Russu, Irina M.

2009-01-01

342

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

PubMed

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

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

2014-01-01

343

The Origins of the RNA World  

PubMed Central

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

Robertson, Michael P; Joyce, Gerald F

2012-01-01

344

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts  

PubMed Central

A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown.

Liu, Yaoquan; Santi, Daniel V.

2000-01-01

345

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

Microsoft Academic Search

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

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

2007-01-01

346

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

PubMed

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

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

2014-06-01

347

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

PubMed Central

Cationic polymers are potential intracellular carriers for small interfering RNA (siRNA). The short and rigid nature of an siRNA chain often results in larger and more loosely packed particles compared to plasmid DNA (pDNA) after complexing with carrier polycations, and in turn, poor silencing effects are seen against the target mRNAs. A helper polyanion, pDNA, was incorporated along with siRNA to form compact nanosized polyplexes. At C/A (cation/anion) ratios of 2 and 5, poly(L-lysine) (PLL)/siRNA-pGFP and PLL/siRNA-pGFP-OSDZ (oligomeric sulfadiazine (OSDZ) for endosomolysis) complexes produced particles 90–150 nm in size with a 15–45 mV surface charge, while PLL/siRNA complexes yielded particles 1–2 ?m in size at the same C/A ratios. The PLL/siRNA-pGFP (C/A 2) complexes showed significantly higher specific gene silencing (50–90% vs. 10–25%) than the complexes formed at C/A 5. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes improved the specific gene silencing (90%) more dramatically than PLL/siRNA-pGFP (C/A 2) complexes (50%), demonstrating a potential role for OSDZ. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes sustained higher specific gene silencing compared with PLL/siRNA-pGFP (C/A 2) complexes. Other oligomeric sulfonamides (OSA) with varying pKa used in PLL/siRNA-pGFP-OSA complexes also caused effective gene silencing. The pGFP in the PLL/siRNA-pGFP complexes successfully expressed GFP protein without interfering with the siRNA. In conclusion, this study demonstrates that long pDNA helps effectively form nanosized siRNA particles and that OSA enhances specific gene silencing. In a single nucleic acid carrier formulation, co-delivery of siRNA and pDNA is feasible to maximize therapeutic effects or to include therapeutic or diagnostic functionalities.

Kang, Han Chang; Bae, You Han

2011-01-01

348

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

PubMed Central

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

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

2010-01-01

349

CorreLogo: an online server for 3D sequence logos of RNA and DNA alignments  

PubMed Central

We present an online server that generates a 3D representation of properties of user-submitted RNA or DNA alignments. The visualized properties are information of single alignment columns, mutual information of two alignment positions as well as the position-specific fraction of gaps. The nucleotide composition of both single columns and column pairs is visualized with the help of color-coded 3D bars labeled with letters. The server generates both VRML and JVX output that can be viewed with a VRML viewer or the JavaView applet, respectively. We show that combining these different features of an alignment into one 3D representation is helpful in identifying correlations between bases and potential RNA and DNA base pairs. Significant known correlations between the tRNA 3? anticodon cardinal nucleotide and the extended anticodon were observed, as were correlations within the amino acid acceptor stem and between the cardinal nucleotide and the acceptor stem. The online server can be accessed using the URL .

Bindewald, Eckart; Schneider, Thomas D.; Shapiro, Bruce A.

2006-01-01

350

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

PubMed

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

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

2013-06-28

351

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

PubMed

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

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

2013-10-18

352

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

PubMed

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

Hatai, Hitoshi; Ochiai, Kenji; Umemura, Takashi

2009-07-01

353

Ultrafast excited-state dynamics of RNA and DNA C tracts  

PubMed Central

The excited-state dynamics of the RNA homopolymer of cytosine and of the 18-mer (dC)18 were studied by steady-state and time-resolved absorption and emission spectroscopy. At pH 6.8, excitation of poly(rC) by a femtosecond UV pump pulse produces excited states that decay up to one order of magnitude more slowly than the excited states formed in the mononucleotide cytidine 5’-monophosphate under the same conditions. Even slower relaxation is observed for the hemiprotonated, self-associated form of poly(rC), which is stable at acidic pH. Transient absorption and time-resolved fluorescence signals for (dC)18 at pH 6.8 are similar to ones observed for poly(rC) near pH 4, indicating that hemiprotonated structures are found in DNA C tracts at neutral pH. In both systems, there is evidence for two kinds of emitting states with lifetimes of ~100 ps and slightly more than 1 ns. The former states are responsible for the bulk of emission from the hemiprotonated structures. Evidence suggests that slow electronic relaxation in these self-complexes is the result of vertical base stacking. The similar signals from RNA and DNA C tracts suggest a common base-stacked structure, which may be identical with that of i-motif DNA.

Cohen, Boiko; Larson, Matthew H.; Kohler, Bern

2008-01-01

354

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

PubMed

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

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

2006-11-01

355

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities  

PubMed Central

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

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

2014-01-01

356

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

PubMed Central

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

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

2012-01-01

357

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

PubMed Central

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

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

1997-01-01

358

MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells.  

PubMed

We examined the role of miRNAs in DNA damage response in HepG2 cells following exposure to 4-aminobiphenyl (4-ABP). The arylamine 4-ABP is a human carcinogen. Using the Comet assay, we showed that 4-ABP (18.75-300?M) induces DNA damage in HepG2 cells after 24h. DNA damage signaling pathway-based PCR arrays were used to investigate expression changes in genes involved in DNA damage response. Results showed down-regulation of 16 DNA repair-related genes in 4-ABP-treated cells. Among them, the expression of selected six genes (UNG, LIG1, EXO1, XRCC2, PCNA, and FANCG) from different DNA repair pathways was decreased with quantitative real-time PCR (qRT-PCR). In parallel, using the miRNA array, we reported that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these differential 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, and was predicted to be implicated in the deregulation of FANCG and RAD18 genes, respectively, via bioinformatic analysis. Both FANCG and RAD18 proteins were found to be down-regulated in 4-ABP-treated cells. In addition, overexpression and knockdown of miRNA-513a-5p and miRNA-630 reduced and increased the expression of FANCG and RAD18 proteins, respectively. Based on the above results, we indicated that miRNA-513a-5p and miRNA-630 could play a role in the suppression of DNA repair genes, and eventually lead to DNA damage. PMID:24857880

Huan, Lin Chen; Wu, Jong-C; Chiou, Bin-Hao; Chen, Chin-Hui; Ma, Nianhan; Chang, Chi Yao; Tsen, Yi-Kuang; Chen, Ssu Ching

2014-08-01

359

tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma  

PubMed Central

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.

Maute, Roy L.; Schneider, Christof; Sumazin, Pavel; Holmes, Antony; Califano, Andrea; Basso, Katia; Dalla-Favera, Riccardo

2013-01-01

360

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

PubMed Central

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

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

2010-01-01

361

Small Interfering RNA (siRNA)-Directed Knockdown of Uracil DNA Glycosylase Induces Apoptosis and Sensitizes Human Prostate Cancer Cells to Genotoxic Stress  

PubMed Central

Uracil DNA glycosylase (UNG) is the primary enzyme responsible for removing uracil residues from DNA. Although a substantial body of evidence suggests that DNA damage plays a role in cancer cell apoptosis, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in cell survival. To test the hypothesis that the repair of DNA damage associated with uracil misincorporation is critical for cancer cell survival, we used small interfering RNA to target the human UNG gene. In a dose- and time-dependent manner, siRNA specifically inhibited UNG expression and modified expression of several genes, at both mRNA and protein levels. In LNCaP cells, p53, p21 and Bax protein levels increased, whereas Bcl2 levels decreased. In DU145 cells, p21 levels were elevated, although mutant p53 and Bax levels remained unchanged. In PC3 cells, UNG inhibition resulted in elevated p21 and Bax levels. In all three cell lines, UNG inhibition reduced cell proliferation, induced apoptosis, and increased cellular sensitivity to genotoxic stress. Furthermore, the in vitro cleavage experiment using uracil-containing double-stranded DNA as template has demonstrated that siRNA-mediated knockdown of UNG expression significantly reduced uracil-excising activity of UNG in human prostate cancer cells, which was associated with DNA damage analyzed by comet assay. Taken together, these findings indicate that RNA interference-directed targeting of UNG is a convenient, novel tool for studying the biological role of UNG and raises the potential of its application for prostate cancer therapy.

Pulukuri, Sai Murali Krishna; Knost, James A.; Estes, Norman; Rao, Jasti S.

2009-01-01

362

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD  

SciTech Connect

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of {alpha}-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.

Nowotny, Marcin; Cerritelli, Susana M.; Ghirlando, Rodolfo; Gaidamakov, Sergei A.; Crouch, Robert J.; Yang, Wei (NIH)

2008-07-09

363

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

PubMed Central

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

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

1981-01-01

364

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.  

PubMed

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb). PMID:8879595

Chenchik, A; Diachenko, L; Moqadam, F; Tarabykin, V; Lukyanov, S; Siebert, P D

1996-09-01

365

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

PubMed

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

Chuang, Trees-Juen; Chiang, Tai-Wei

2014-01-01

366

Systematic Functional Comparative Analysis of Four Single-Stranded DNA-Binding Proteins and Their Affection on Viral RNA Metabolism  

PubMed Central

The accumulation of single-stranded DNA-binding (SSB) proteins is essential for organisms and has various applications. However, no study has simultaneously and systematically compared the characteristics of SSB proteins. In addition, SSB proteins may bind RNA and play an unknown biological role in RNA metabolism. Here, we expressed a novel species of SSB protein derived from Thermococcus kodakarensis KOD1 (KOD), as well as SSB proteins from Thermus thermophilus (TTH), Escherichia coli, and Sulfolobus Solfataricus P2 (SSOB), abbreviated kod, tth, bl21, and ssob, respectively. These SSB proteins could bind ssDNA and viral RNA. bl21 resisted heat treatment for more than 9 h, Ssob and kod could withstand 95°C for 10 h and retain its ssDNA- and RNA-binding ability. Four SSB proteins promoted the specificity of the DNA polymerase in PCR-based 5- and 9-kb genome fragment amplification. kod also increased the amplification of a 13-kb PCR product, and SSB protein–bound RNA resisted Benzonase digestion. The SSB proteins could also enter the host cell bound to RNA, which resulted in modulation of viral RNA metabolism, particularly ssob and bl21.

Guo, Jinlei; Zhang, Xun; Song, Haiyan; Lv, Jianxin; Gao, Jimin; Wang, Yuepeng; Chen, Litian; Wang, Yue

2013-01-01

367

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

PubMed Central

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

Chuang, Trees-Juen; Chiang, Tai-Wei

2014-01-01

368

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

PubMed Central

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

Hoffman, David J.; Niyogi, Salil K.

1973-01-01

369

Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana.  

PubMed

RNA-directed DNA methylation (RdDM) is an epigenetic process whereby small interfering RNAs (siRNAs) guide cytosine methylation of homologous DNA sequences. RdDM requires two specialized RNA polymerases: Pol IV transcribes the siRNA precursor whereas Pol V generates scaffold RNAs that interact with siRNAs and attract the methylation machinery. Recent evidence also suggests the involvement of RNA polymerase II (Pol II) in recruiting Pol IV and Pol V to low copy, intergenic loci. We demonstrated previously that Pol V-mediated methylation at a transgene locus in Arabidopsis spreads downstream of the originally targeted region by means of Pol IV/RNA-DEPENDENT RNA POLYMERASE2 (RDR2)-dependent 24-nt secondary siRNAs. Here we show that these secondary siRNAs can not only induce methylation in cis but also in trans at an unlinked target site, provided this sequence is transcribed by Pol II to produce a non-coding RNA. The Pol II transcript appears to be important for amplification of siRNAs at the unlinked target site because its presence correlates not only with methylation but also with elevated levels of 24-nt siRNAs. Potential target sites that lack an overlapping Pol II transcript and remain unmethylated in the presence of trans-acting 24-nt siRNAs can nevertheless acquire methylation in the presence of 21-24-nt hairpin-derived siRNAs, suggesting that RdDM of non-transcribed target sequences requires multiple size classes of siRNA. Our findings demonstrate that Pol II transcripts are not always needed for RdDM at low copy loci but they may intensify RdDM by facilitating amplification of Pol IV-dependent siRNAs at the DNA target site. PMID:23512103

You, Wanhui; Lorkovic, Zdravko J; Matzke, Antonius J M; Matzke, Marjori

2013-05-01

370

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

SciTech Connect

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.

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

1985-02-01

371

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

PubMed

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

Wei, Zewen; Li, Zhihong

2014-01-01

372

Simplified Identification of mRNA or DNA in Whole Cells  

NASA Technical Reports Server (NTRS)

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

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

373

Quantitative PCR for detection of mRNA and gDNA in environmental isolates.  

PubMed

Quantitative PCR is used to gauge the abundance of specific nucleic acid species within purified samples. Due to its high sensitivity and minimal operation costs, this method is routinely applied in modern molecular bioscience laboratories. Nonetheless, all quantitative PCR experiments must include several carefully designed, yet simple, controls to ensure the reliability of the analyses. The aim of this chapter is to provide basic quantitative PCR methods, from primer design through data analysis, that are generally applicable to studies in microbiology. These methods allow the abundance of targeted RNA or DNA molecules to be determined in nucleic acid samples purified from a variety of biological sources. PMID:24515358

Brzoska, Anthony J; Hassan, Karl A

2014-01-01

374

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

NASA Astrophysics Data System (ADS)

The photoelectron-photoion-photoion coincidence method is used to measure the photodissociation of doubly charged D-ribose (C5H10O5), the RNA sugar molecules, and 2-deoxy-D-ribose (C5H10O4), the DNA sugar molecules, following normal Auger decay after initial C 1s and O 1s core ionizations. The fragment identification is facilitated by measuring isotopically labeled D-ribose, such as D-ribose deuterated at C(1), and with 13C at the C(5) position. Ab initio quantum chemistry calculations are used to gain further insight into the abundant appearance of the CHO+ fragment.

Ha, D. T.; Huels, M. A.; Huttula, M.; Urpelainen, S.; Kukk, E.

2011-09-01

375

DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy  

PubMed Central

Multifunctional DNA micelles: Molecular beacon micelle flares (MBMFs), based on diacyllipid-molecular beacon conjugate (L-MB) self-assembly, have been developed for combined mRNA detection and gene therapy. The advantages of these micelle flares include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high signal-to-background ratio, excellent target selectivity, and superior biocompatibility. In addition, these probes possess a hydrophobic cavity that can be used for additional hydrophobic agents, holding great promise for constructing an all-in-one nucleic acid probe.

Chen, Tao; Sam Wu, Cuichen; Jimenez, Elizabeth; Zhu, Zhi; Dajac, Joshua G.; You, Mingxu; Han, Da

2013-01-01

376

Nucleotide Sequence of an RNA Polymerase Binding Site from the DNA of Bacteriophage fd  

Microsoft Academic Search

The primary structure of a strong RNA polymerase binding site in the replicative form DNA of phage fd has been determined by direct DNA sequencing. It is: 5' T-G-C-T-T-C-T-G-A-C-T-A-T-A-A-T-A-G-A-C-A-G-G-G-T-A-A- 3' A-C-G-A-A-G-A-C-T-G-A-T-A-T-T-A-T-C-T-G-T-C-C-C-A-T-T- A-G-A-C-C-T-G-A-T-T-T-T-T-G-A 3' T-C-T-G-G-A-C-T-A-A-A 5' the molecule contains regions with 2-fold symmetry and sequence homologies to promoter regions from other DNAs. The startpoint of transcription is located in the center

Heinz Schaller; Christopher Gray; Karin Herrmann

1975-01-01

377

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

NASA Astrophysics Data System (ADS)

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

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

2008-03-01

378

Non-covalent interactions: complexes of guanidinium with DNA and RNA nucleobases.  

PubMed

Considering that guanidine-based derivatives are good DNA minor groove binders, we have theoretically studied, using the Polarizable Continuum model mimicking water solvation, the complexes formed by the biologically relevant guanidinium cation and the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil). The interactions established within these complexes both by hydrogen bonds and by cation-? interactions have been analyzed by means of the Atoms in Molecules and Natural Bond Orbital approaches. Moreover, maps of electron density difference have been produced to understand the cation-? complexes. Finally, the NICS and three-dimensional NICS maps of the cation-? complexes have been studied to understand the effect of the guanidinium cation on the aromaticity of the nucleobases. PMID:23992551

Blanco, Fernando; Kelly, Brendan; Sánchez-Sanz, Goar; Trujillo, Cristina; Alkorta, Ibon; Elguero, Jose; Rozas, Isabel

2013-10-01

379

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

PubMed

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

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

2012-02-14

380

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

PubMed Central

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

Braddock, M; Hardie, D G

1988-01-01

381

Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells.  

PubMed

Gene silencing mediated by double-stranded RNA is a sequence-specific RNA degradation mechanism highly conserved in eukaryotes that serves as an antiviral defense pathway in both plants and Drosophila. Short interfering RNAs (siRNAs), the 21- to 23-nt double-stranded intermediates of this natural defense mechanism, are becoming powerful tools for reducing gene expression and countering viral infection in a variety of mammalian cells. Here we report the use of siRNAs to target reporter gene expression and viral DNA accumulation in cultured plant cells. Transient expression of reporter genes encoding either GFP or red fluorescent protein from Discosoma was specifically reduced by 58% and 47%, respectively, at 24 h after codelivery of cognate siRNAs in BY2 protoplasts. In contrast to mammalian systems, the siRNA-induced silencing of GFP expression was transitive as indicated by the presence of siRNAs representing parts of the target RNA outside the region homologous to the triggering siRNA. Codelivery of an siRNA designed to target the mRNA encoding the replication-associated protein (AC1) of the geminivirus African cassava mosaic virus (ACMV) from Cameroon blocked AC1 mRNA accumulation by approximately 91% and inhibited accumulation of the ACMV genomic DNA by approximately 66% at 36 and 48 h after transfection. As with siRNA-induced reporter gene silencing, the siRNA targeting ACMV AC1 was specific and did not affect the replication of East African cassava mosaic Cameroon virus. This report demonstrates the occurrence of siRNA-mediated suppression of gene expression in cultured plant cells and that siRNA can interfere with and suppress accumulation of a nuclear-replicated DNA virus. PMID:12886005

Vanitharani, Ramachandran; Chellappan, Padmanabhan; Fauquet, Claude M

2003-08-01

382

Characterization of RNA aptamers that disrupt the RUNX1-CBF?/DNA complex  

PubMed Central

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor ? protein (CBF?). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD–CBF? complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity (?100–160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBF?, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD–CBF? complex and thus will further aid exploring the functions of this key transcription factor.

Barton, Jenny L.; Bunka, David H. J.; Knowling, Stuart E.; Lefevre, Pascal; Warren, Alan J.; Bonifer, Constanze; Stockley, Peter G.

2009-01-01

383

Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon".  

PubMed Central

The efficiency of translation depends on correct tRNA-ribosome interactions. The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications. Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop. Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding. To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops. The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe). However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding. Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding.

Dao, V; Guenther, R; Malkiewicz, A; Nawrot, B; Sochacka, E; Kraszewski, A; Jankowska, J; Everett, K; Agris, P F

1994-01-01

384

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.  

PubMed Central

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

385

Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules  

Microsoft Academic Search

Oligonucleotides containing 5-(N-aminohexyl) carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides con- taining 5-(N-aminohexyl)carbamoyl-20-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-20-O-methyl- uridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA

E. C. M. Juan; J. Kondo; T. Kurihara; T. Ito; Y. Ueno; A. Matsuda; A. Takenaka

2007-01-01

386

Production andCharacterization ofAnti-DNA-RNA Monoclonal Antibodies andTheirApplication in Listeria Detection  

Microsoft Academic Search

Twodifferent immunoglobulin M MAbswere isolated. The20D3MAb,generated withthe+X174 DNA-RNAhybrid, showedassociation constants of1.05 x 1o12, 2.12x 1010, and1.68x 107fortheantigens +X174 DNA-RNA,cDNA-mRNA,andpoly(rA)-poly(dT), respectively. The6B5MAb,obtained withthecDNA-mRNAhybrid, showedassociation constants of1.59x l0S, 5 x 1012, and7.1x 108fortheabove-described antigens, respectively. Withthe20D3MAb,an immunoassay was developed forthedetection ofListeria DNA-RNAhybrids. Inbrief, abiotinylated rRNAgene probespecific forthegenusListeria was hybridized withrRNAinthesolution phase. Thehybrids thusformed were thencaptured inmicrotiter plate wells precoated withthepurified 20D3MAb,andtheprobe-target hybrids

ISMAIL FLISS; E. EMOND; R. LEMIEUX; R. E. SIMARD; A. ET; S. PANDIAN

1993-01-01

387

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

PubMed Central

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

Arima, Hidetoshi; Motoyama, Keiichi; Higashi, Taishi

2012-01-01

388

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

PubMed

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

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

2014-05-01

389

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

PubMed Central

Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ?18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spo