Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene (which stands for adenosine deaminase acting on RNA). Adenosine deaminases acting on RNA (ADAR) are enzymes responsible for binding to double stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) by deamination. ADAR protein is a RNA-binding protein, which functions in RNA-editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of the RNA. The conversion from A to I in the RNA disrupt the normal A:U pairing which makes the RNA unstable. Inosine is structurally similar to that of guanine (G) which leads to I to cytosine (C) binding. In RNA I functions the same as G in both translation and replication. Codon changes can arise from editing which may lead to changes in the coding sequences for proteins and their functions. Most editing site are found in noncoding regions of RNA such as untranslated regions (UTRs), Alu elements and long ...
TY - JOUR. T1 - Identification of human autoantibodies to the DNA ligase IV/XRCC4 complex and mapping of an autoimmune epitope to a potential regulatory region. AU - Lee, Kyung Jong. AU - Dong, Xingwen. AU - Wang, Jingsong. AU - Takeda, Yoshihiko. AU - Dynan, William S.. PY - 2002/9/15. Y1 - 2002/9/15. N2 - The nonhomologous end-joining pathway is the principal mechanism for repair of ionizing radiation-induced, double-strand breaks in mammalian cells. Three polypeptides in this pathway, including the two subunits of Ku protein and the catalytic subunit of the DNA-dependent protein kinase, are known targets of autoantibodies in systemic rheumatic diseases. Here we show that two additional polypeptides in the pathway, DNA ligase IV and XRCC4, are also targets of autoantibodies. These Abs were present in 20% of patients with systemic lupus erythematosus and overlap syndrome. Previous work has shown that XRCC4 is subject to radiation-induced post-translational modification, including ...

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal ...
TY - JOUR. T1 - Sieve-based device for MALDI sample preparation. I. Influence of sample deposition conditions in oligonucleotide analysis to achieve significant increases in both sensitivity and resolution. AU - Molin, Laura. AU - Cristoni, Simone. AU - Crotti, Sara. AU - Bernardi, Luigi Rossi. AU - Seraglia, Roberta. AU - Traldi, Pietro. PY - 2008/11. Y1 - 2008/11. N2 - Spraying of oligonucleotide-matrix solutions through a stainless steel (ss) sieve (38 μm, 450 mesh) leads to the formation, on the matrix-assisted laser desorption/ionization (MALDI) sample holder, of uniformly distributed microcrystals, well separated from each other. When the resulting sample holder surface is irradiated by laser, abundant molecular species form, with a clear increase in both intensity and resolution with respect to values obtained by Dried Droplet, Double Layer, and Sandwich deposition methods. In addition, unlike the usual situation, the sample is perfectly homogeneous, and identical spectra are ...
A polynucleotide molecule is a biopolymer composed of 13 or more nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with distinct biological function. The prefix poly comes from the ancient Greek πολυς (polys, many). DNA consists of two chains of polynucleotides, with each chain in the form of a helical spiral. Although DNA and RNA do not generally occur in the same polynucleotide, the four species of nucleotides may occur in any order in the chain. The sequence of DNA or RNA species for a given polynucleotide is the main factor determining its function in a living organism or a scientific experiment. Polynucleotides occur naturally in all living organisms. The genome of an organism consists of complementary pairs of enormously long polynucleotides wound around each other in the form of a double helix. Polynucleotides have a variety of other roles in organisms. Polynucleotides are used in biochemical ...
DNA bases can be modified by endogenous agents (e.g. oxidized by products of respiration and photosynthesis or methylated by gene silencing processes) as well as by environmental agents (e.g. oxidized by UV light). In the process of removing modified bases, a 3-phosphate group is sometimes left in the resulting gap, and has to be removed since it blocks the incorporation of a new nucleotide by DNA polymerase. The aim of this thesis was the characterization of AtZDP, a plant enzyme with a DNA 3-phosphatase activity.. By homologous modeling, the existence of four domains was predicted in AtZDP, three independent zinc-finger and one DNA 3-phosphatase domains. AtZDP was found to be localized in the nucleus by bimolecular fluorescence complementation. Western blotting analysis showed that the enzyme was ubiquitously expressed in plant tissues.. AtZDP was found in a 600,000 molecular-weight protein complex by gel chromatography and glycerol gradient sedimentation centrifugation. The fractions ...
BioAssay record AID 197831 submitted by ChEMBL: Perfect off-state by site-specific incorporation of o-nitrobenzyl (o-NB) unit into RNase S was determined before photolysis at 10 x E5 M/min.
Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is composed of two main components: hTERT and hTERC. hTERT seems to be the rate-limiting factor for telomerase activity, although hTERC expression was also shown to correlate to a certain extent with telomerase reactivation. To determine whether the absence of hTERC expression could be the consequence of DNA methylation, we quantified hTERC RNA in 60 human samples (19 telomerase-negative normal tissues, nine telomerase-positive and 22 telomerase-negative tumor tissues, eight telomerase-positive and two telomerase-negative cell lines) using a quantitative dot blot on RT-PCR products. Most of the normal tissues did not express hTERC whereas, in telomerase-positive cell lines and in telomerase-positive tumor tissues, a strong up-regulation was observed, suggesting that hTERC transcription is up-regulated during tumorigenesis. The two telomerase-negative cell lines did not express hTERC. In a series of 22 telomerase-negative ...
This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Extracellular RNA may circulate as non-bound RNA, protein-bound RNA, lipid-RNA complexes, lipoprotein (proteolipid)-RNA complexes, protein-RNA complexes including within or in association with ribonucleoprotein complexes, nucleosomes, or within apoptotic bodies. Any intracellular RNA found in plasma or serum can additionally be detected by this invention. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor
0111] In a preferred embodiment, the invention provides for a method of in vitro recombination comprising: [0112] (1) obtaining modified polynucleotide fragments according any one of the method described in any one of claims 1 to 5; [0113] (2) screening some or all of said modified polynucleotides to determine which polynucleotide or polynucleotides encode a protein or proteins of interest; [0114] (3) digesting said modified polynucleotides encoding a protein or proteins of interest with restriction enzymes to form fragments with single-stranded overhangs consisting of three nucleotide residues or of nucleotide residues in multiples of three; [0115] (4) modifying the obtained polynucleotide fragments of step (3) by removing and/or filling in the single-stranded overhangs to obtain new modified fragments by [0116] (i) removing, in multiples of three, all of the nucleotide residues of said overhanging end of one or more polynucleotide fragments; or [0117] (ii) extending the single strand of the ...
1. Remove the 10X T4 DNA Ligase Reaction Buffer* from the freezer to thaw. You can remove the T4 DNA Ligase enzyme from the freezer at this point but leave the ligase in a cold box to keep it close to -20○ C. Thawing is fast if the buffer tube is immersed in room temperature water. Once thawed, agitate the 10X T4 DNA Ligase Reaction Buffer until all precipitate goes into solution. 2. Add 11ul of H2O to a 200ul PCR tube. 3. Add 2ul from each of the digest to the tube.** 4. Add 2ul of 10X T4 DNA Ligase Reaction Buffer to the tube. 5. Add 1ul of the T4 DNA Ligase to the tube. 6. The total volume in each tube should now be 20ul. Ensue the ligation is well mixed by flicking the tube. You can spin the tube in a microcentrifuge for a few seconds to collect the liquid in the bottom of the tube again. *Repeated freeze-thaw cycles of the buffer can degrade the ATP in the buffer thereby making the ligation reaction less efficient. It is wise to aliquot the buffer into 10ul aliquots prior to freezing ...
Small RNA libraries from Xenopus microtubules and egg extracts were prepared for sequencing on the Genome Sequencer FLX system as previously described by Lau et al (2006). To minimize sample loss in small RNA library construction from single eggs, we modified the library construction procedure as follows. Total RNAs were extracted from single eggs using TRI Reagent (∼0.1-1 μg), and a pre‐adenylated 3′ linker sequence designed for an Illumina GA‐II sequencer flowcell was ligated to the total RNA using T4 RNA ligase. The 3′ linker ligation reactions were then resolved on a 15% denaturing polyacrylamide gel alongside 18 nt and 31 nt RNA markers that were also marked with the 3′ linker. A 5′linker was ligated to purified 3′ ligation products overnight at 16°C using T4 RNA ligase. The 5′ ligation products were then directly subjected to reverse transcription and limited (,15) cycles of PCR, and the PCR products representing the small RNA population was gel purified, and this ...
Polyadenylated (poly(A)+) RNA molecules have been isolated from Methanococcus vannielii. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate (3)Huridine for 3 min at 37C was poly(A)+ RNA. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. Polyadenylate (poly(A)) tracts were isolated by digestion with RNase A and RNase T1 after 3 end labeling of the poly(A)+ RNA with RNA ligase. The radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5 termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate ...
0041]This invention relates to methods for detecting tumor-associated RNA in plasma, serum and other bodily fluids. The methods thereby provide for the detecting, diagnosing, inferring, evaluating or monitoring of cancer or neoplastic disease in a human or animal. The inventive methods comprise the steps of extracting RNA from plasma, serum, or bodily fluid of a human or animal, and thereafter assessing the amount or concentration of mammalian extracellular RNA in said plasma, serum, or other bodily fluid of the human or animal, or cDNA derived therefrom. Particularly preferred embodiments of the inventive steps include an amplification or signal amplification step, followed by detection of the amplified product or signal. In particularly preferred embodiments this is performed by comparing the amount or concentration of one or more RNA species in said plasma, serum, or bodily fluid obtained from the human or animal with another, or with the amount or concentration of RNA found in bodily fluid ...
COMPOSITIONS AND METHODS FOR CANCER AND CANCER STEM CELL DETECTION AND ELIMINATION - In alternative embodiments, the invention provides compositions and methods for inhibiting or ablating cancer stem cells. In alternative embodiments, the invention provides compositions and methods for inhibiting the action of double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes. In alternative embodiments, the invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML). In alternative embodiments, the invention provides compositions and methods for inhibiting a Sonic Hedgehog (Shh) pathway, e.g., by using a Smoothened (SMO) protein inhibitor. In alternative embodiments, the invention provides compositions and methods for ...
Abstract A series of 16 yellow fever (YF) viruses isolated from mosquitoes, monkeys and humans in different epidemiological contexts in Senegal and The Gambia between 1976 and 1983, was analyzed by T1 RNase oligonucleotide fingerprints of the genomic 32P-labeled RNA, by SDS-polyacrylamide gel electrophoresis of the intracellular virus-specified polypeptides, by peptide mapping of the envelope E glycoprotein and by immunological reactivities with monoclonal antibody fluids (MAF's) against the E glycoprotein. These strains had not been passed in suckling mice and were isolated in Aedes pseudoscutellaris Mos 61 cultured cells. These strains showed no virulence in three-week-old Swiss mice when injected intraperitoneally. Direct comparison of the large T1 RNase-resistant oligonucleotide maps indicated a relative genetic stability (92%-100%). A greater change was observed when these strains were compared with an epidemic YF strain isolated in 1965 with an oligonucleotide fingerprint map sharing 82%-88%
Maximiliano DAngelo, PhD, Research Associate, Salk Institute for Biological Studies: Nuclear Pore Deterioration during Aging and Its Consequences for Cellular Function. Kristian Doyle, PhD, Post-Doctoral Scholar, Stanford University: Does Increased TGF-Beta Signaling in the Elderly Increase Astrogliosis and Impair Functional Recovery Following Stroke?. Jeremy Herskowitz, PhD, Postdoctoral Fellow, Emory University School of Medicine: The Role of LR11 in Alzheimers Disease Pathogenesis. Maria Lehtinen, PhD, Postdoctoral Fellow, HHMI/Childrens Hospital Boston: Regulation of the Neural Stem Cell Niche by the Aging Cerebrospinal Fluid (CSF) Proteome. Lingbo Li, PhD, Stanford University: Understanding the Role of RNA-editing Enzyme ADAR in Aging Neurons. Daijun Ling, PhD, Research Fellow, Beckman Research Institute of City of Hope: Autophagic-Lysosomal Storage of Amyloid Beta and Its Connection to Development of Senile Plaques. Brian Onken, PhD, Postdoctoral Fellow, Rutgers, The State University of ...
RNALogo: a new approach to display structural RNA alignment - Regulatory RNAs play essential roles in many essential biological processes, ranging from gene regulation to protein synthesis. This work presents a web-based tool, RNALogo, to create a new graphical representation of the patterns in a multiple RNA sequence alignment with a consensus structure. The RNALogo graph can indicate significant features within an RNA sequence alignment and its consensus RNA secondary structure. RNALogo extends Sequence logos, and specifically incorporates RNA secondary structures and mutual information of base-paired regions into the graphical representation. Each RNALogo graph is composed of stacks of letters, with one stack for each position in the consensus RNA secondary structure. RNALogo provides a convenient and high configurable logo generator. An RNALogo graph is generated for each RNA family in Rfam, and these generated logos are accumulated into a gallery of RNALogo. Users can search or browse RNALogo
Analusis, a European journal on Analytical Chemistry, is published under the auspices of the Société Française de Chimie, the Société de Chimie Industrielle and the Gesellschaft Deutscher Chemiker
TY - JOUR. T1 - Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis. AU - Repass, John F.. AU - Makino, Shinji. PY - 1998/10. Y1 - 1998/10. N2 - The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5 end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963-4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not ...
Summary Isolates of yellow fever (YF) virus from Africa (Senegambia, Central African Republic, Ivory Coast, Burkina Faso) and from South America (Panama, Ecuador, Trinidad) were examined by oligonucleotide fingerprinting of the 40S genome RNA. Geographically isolated and epidemiologically unrelated viruses were very distinct. On the basis of the T1 oligonucleotide fingerprints of each isolate, four geographical variants (topotypes) of YF virus isolated within the same period of time have been established. The Ivory Coast and Burkina Faso topotypes were similar. In the Central African Republic, two variants could be found exhibiting 70 to 75% homology to one another. In South America, the three analysed strains exhibited only about 70% homology, but could be classified in the same topotype. The oligonucleotide fingerprints of the genome RNA offered a useful tool for the understanding of YF virus variability.
Understanding how biomolecules interact is a major task of systems biology. To model protein-nucleic acid interactions, it is important to identify the DNA or RNA-binding residues in proteins. Protein sequence features, including the biochemical property of amino acids and evolutionary information in terms of position-specific scoring matrix (PSSM), have been used for DNA or RNA-binding site prediction. However, PSSM is rather designed for PSI-BLAST searches, and it may not contain all the evolutionary information for modelling DNA or RNA-binding sites in protein sequences. In the present study, several new descriptors of evolutionary information have been developed and evaluated for sequence-based prediction of DNA and RNA-binding residues using support vector machines (SVMs). The new descriptors were shown to improve classifier performance. Interestingly, the best classifiers were obtained by combining the new descriptors and PSSM, suggesting that they captured different aspects of evolutionary
Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd ...
A negative-sense single-stranded RNA virus (or (-)ssRNA virus) is a virus that uses negative sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The negative viral RNA is complementary to the mRNA and must be converted to a positive RNA by RNA polymerase before translation. Therefore, the purified RNA of a negative sense virus is not infectious by itself, as it needs to be converted to a positive sense RNA for replication. These viruses belong to Group V on the Baltimore classification. In addition, negative-sense single-stranded RNA viruses have complex genomic sequences, cell cycles, and replication habits that use various protein complexes to arrange in specific conformations and carry out necessary processes for survival and reproduction of their genomic sequences. The complexity of negative-sense single-stranded RNA viruses carries into its ability to suppress the innate immune ...
299057642 - EP 0785941 B1 2000-04-19 - PORPHYRIN LABELING OF POLYNUCLEOTIDES - [origin: WO9611937A1] The invention provides various porphyrin labeled compounds and reagents for labeling molecules with porphyrins. The porphyrin labeled compounds include porphyrin labeled nucleosides, nucleotides, polynucleotides and the like. The porphyrin labeled compounds of the invention are designed so that the porphyrin moiety on the labeled compound may be detected on the basis of a reaction product produced by the porphyrin catalyzed oxidation of a porphyrin detection reagent. The oxidation of the porphyrin detection reagent may result in the formation of light, i.e., a chemiluminescent reaction, or a colored compound, i.e., a colorimetric reaction. The compounds of the invention contain the general structure of: porphyrin-linker-base-sugar-(phos)n-OH. Another aspect of the invention is to provide methods of labeling and detecting polynucleotides by incorporating a porphyrin-labeled nucleotide into a
Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer using carbohydrates as the feedstock [1]. Therefore, it is important to develop molecular biology tools to further manipulate this microorganism. Extraction of high-quality RNA from R. toruloides is particular challenging due to high level of polysaccharides, lipids and other secondary metabolites [2]. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods, including guanidine thiocyanate [3], glass beads-hot acid phenol, glass beads-TRIzol, and modified RNAiso, were evaluated. RNA quality was assessed using UV absorbance (A260/A280 and A260/A230), agarose gel electrophoresis, reverse transcription (RT)-PCR reactions, and Gel-Pro analyzer 4.5. Large differences in RNA yield and quality among protocols were found. The optimum method was modified RNAiso method, where RNA was isolated using liquid nitrogen-RNAiso with salt precipitation and the addition of PVP andβ-mercaptoethanol. This ...
Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascula...
E. coli DNA Ligase catalyzes the formation of phosphodiester bonds between double-stranded DNA fragments containing juxtaposed 5-phosphate termini and 3-hydroxyl termini in the presence of the NAD cofactor. Both T4 DNA Ligase and E.coli DNA Ligase are used in various gene cloning experiments; however, unlike T4 DNA Ligase, E. coli DNA Ligase can only catalyze ligation of cohesive end-containing DNA fragments via standard reaction conditions. E. coli DNA Ligase is supplied in a buffer containing 10 mM potassium phosphate (pH 7.5), 50 mM KCl, 1 mM DTT, 1 mM EDTA and 50% glycerol.. ...
Tan,M.H., Li,Q., Shanmugam,R., Piskol,R., Kohler, J., et al. (2017) Dynamic landscape and regulation of RNA editing in mammals. Nature 550(7675):249-254. Ma, D, George C.X., Nomburg, J., Pfaller, C.K., Cattaneo, R., and Samuel, C.E. (2017). Upon infection the cellular WD repeat-containing protein 5 localizes to cytoplasmic inclusion bodies and enhances measles virus replication. J. Virol.: in press. George, C.X., Ramaswami, G., Li, J.B., Samuel, C.E. (2016) Editing of cellular self-RNAs by adenosine deaminase ADAR1 suppresses innate immune stress responses. J Biol Chem. 291:6158-68. Kainulainen, M., Lau, S., Samuel, C.E., Hornung, V., Weber, F. (2016) NSs virulence factor of Rift Valley Fever Virus engages the F-box proteins FBXW11 and Î²-TRCP1 to degrade the antiviral protein kinase PKR. J Virol. 90:6140-6147. George, C. X., Samuel, C. E. (2015) STAT2-dependent induction of RNA adenosine deaminse ADAR1 by type 1 interferon differs between mouse and human cells in the requirement for STAT1. ...
Poly(ADP-ribose) polymerase (PARP) is a critical DNA repair enzyme involved in DNA single-strand break repair via the base excision repair pathway. PARP inhibitors have been shown to sensitize tumors to DNA-damaging agents and to also selectively kill homologous recombination repair-defective cancers, such as those arising in BRCA1 and BRCA2 mutation carriers. Recent proof-of-concept clinical studies have demonstrated the safety and substantial antitumor activity of the PARP inhibitor, olaparib in BRCA1/2 mutation carriers, highlighting the wide therapeutic window that can be achieved with this synthetic lethal strategy. Likewise, the PARP inhibitor, BSI-201, in combination with carboplatin and gemcitabine have produced promising results in "triple-negative" breast cancers. There are also currently numerous other PARP inhibitors in clinical development. The potential broader therapeutic application of these approaches to a wide range of sporadic tumors harboring specific defects in the ...
Purpose: The strongest genetic association of Fuchs endothelial corneal dystrophy (FECD) is with an expanded trinucleotide repeat (CTG·CAG) in an intron of the TCF4 gene. The same expanded repeat sequence in the 3UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1) via a RNA toxicity mechanism. In DM1, poly(CUG) transcripts accumulate in foci and sequester the splicing factor MBNL1, causing missplicing of essential transcripts in skeletal muscle. Our hypothesis is that the same molecular mechanism causes RNA toxicity in the corneal endothelium of FECD patients. Because FECD patients do not present signs or symptoms of DM1, we also ask whether the molecular signature of RNA toxicity is present in muscle cells derived from FECD patients.. Methods: Corneal endothelial tissue was obtained at the time of endothelial keratoplasty, and skin fibroblasts were derived from skin biopsy specimens from patients with FECD. Using fluorescence in situ hybridization (FISH), protein-RNA aggregates can be ...
Gene expression is tightly regulated at the level of transcription through cooperation between cis-regulatory elements and trans-factors that bind to the regulatory elements. Together, these factors regulate the higher order chromatin structure which establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and ...
Activation-induced deaminase (AID) is expressed only in germinal center B cells. There, it is required for somatic hypermutation, gene conversion and class switch recombination of antibody variable region segments, three processes that diversify antibodies during immune responses. Although AID has homology to RNA-editing enzymes, three recent reports suggest it could initiate the diversification processes by deaminating cytidine residues within the antibody genes themselves.
The particle-bound RNA polymerase activity of vesicular stomatitis virus (VSV) can be demonstrated in vivo. Linear synthesis of viral RNA persists for 5 to 6 hours at 34°C in infected monolayers of chick embryo cells treated with cycloheximide and actinomycin D to block synthesis of protein and cell-specific RNA. At least 55 percent of the RNA made under these conditions is complementary to virion RNA. RNA synthesis mediated by VSV polymerase activity is inhibited in cells first treated with chick-derived interferon or polyriboinosinate• polyribocytidylate, but not by mouse interferon. The RNA product of VSV polymerase activity is present throughout the cytoplasm, and its synthesis is inhibited by the interferon system, as judged by autoradiographs that show the physical distribution, in cells, of RNA produced by virion polymerase in the absence of translation-a demonstration of the transcription product of the viral genome. ...
The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
Three IgM monoclonal anti-DNA antibodies were produced by hybridoma techniques from an MRL-lpr/lpr mouse using denatured DNA (dDNA) as the selection antigen. All three antibodies also bound poly(dT), poly(rA), and the single-stranded random copolymer poly(dI,dT), and each antibody displayed a unique preference for a limited array of other ribo- and deoxyribopolynucleotides based on direct binding as well as inhibition studies. Inability to identify a common primary structure in the polynucleotides reactive with each antibody suggested that higher ordered structures may be important. This notion was supported by the finding that oligomers of thymidine of 25-30 nucleotides or less were ineffective in blocking antibody binding to dDNA or poly(dT). However, deliberate destabilization of putative secondary structures by decreasing counterion concentration and increasing temperature had little effect on antibody binding to poly(dT). Since the antigenic polynucleotides in general contain little known ...
H-phosphonate chemistry is of value when the internucleotide linakge required is other than the standard phosphodiester linkage. The H-phosphonate monomers shown below are used instead of the phosphoramidite bases. Using this method, the monomer that is able to be activated is a 5-DMT-base-protected, nucleoside 3-hydrogen phosphonate. The presence of the H- phosphonate moiety on these monomers renders phosphate protection unnecessary. The same base protecting groups are used in phosphite triester chemistry. The H-phosphate synthesis cycle is very similar to that of the phosphoramidite method. Slight differences result from the properties of the monomers utilized. For instance, a different activating agent is used. In addition, the H-phosphonate diesteres generated by the coupling reactions are stable to the normal reaction conditions, so oxidation at every step is unnecessary. Instead, a single oxidation step can be performed at the end of the chain elongation. This single oxidation step makes ...
The latest issue of Analyst is now available for you to browse, and we head east for all three covers.. Theres nanoparticle-assisted laser desorption/ionisation MS from Japan, electrochemical biosensing from Korea, and microextraction from Taiwan.. On the front cover is work from Shu Taira of the School of Material Science at the Japan Advanced Institute of Science and Technology (JAIST), Ishikawa, Japan, and colleagues.. They analysed oligonucleotides by nanoparticle-assisted laser desorption/ionization (nano-PALDI) mass spectrometry, and demonstrated that iron-based nanoparticles may serve as the assisting material of ionization for genes and other biomolecules.. Communication: Oligonucleotide analysis by nanoparticle-assisted laser desorption/ionization mass spectrometry ...
Abstract:. The dissemination of AAC(6)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6)-Ib, and the transformant strains were ...
0007] Various embodiments include one or more of the following features: [0008] a ligase reaction buffer contains a small molecule enhancer having a concentration of 1%-50% v/v that is capable of enhancing, by at least 25%, intramolecular or intermolecular ligation of a polynucleotide fragment or fragments than would otherwise be obtained in the absence of the enhancer; [0009] the one or more small molecule ligation enhancers are selected from an optionally substituted straight or branched chain diol or polyol containing 2 to 20 carbons, alcohols, zwitterionic compounds and polar aprotic molecules; [0010] the optionally substituted straight or branched diol or polyol is selected from the group consisting of 1,2-ethylene glycol, 1,2-propanediol (1,2-PrD), 1,3-propanediol (1,3-PrD), glycerol, pentaerythritol, sorbitol, diethylene glycol, dipropyleneglycol, neopentyl glycol, 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, 1,3-butanediol, and 1,4-butanediol 1,5-pentanediol, 1,6-hexanediol, ...
Abstract: Infection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1-24). We identified the low frequency haplotype AACCAT significantly associated with recurrent ...
Escherichia coli polynucleotide phosphorylase (PNPase) is generally believed to be involved in mRNA degradation via its 3$\sp\prime$ to 5$\sp\prime$ exoribonuclease activity. However, there are many unexplained observations related to the physiological role of this enzyme. The projects conducted in this thesis are aimed at understanding more about the in vivo function of PNPase. Two approaches were employed to try to achieve this goal. First, a biochemical approach involving the purification of PNPase associated proteins was used. Specifically, the previously documented $\beta$ subunit present in the pentameric form of PNPase was examined as an initial step toward understanding the mechanism and possibly the regulation of PNPase catalysis. Second, a genetic approach was undertaken which focused on characterization of mutant strains lacking PNPase in combination with other known enzymes involved in RNA metabolism, in particular, tRNA nucleotidyltransferase, poly(A) polymerase and RNase PH.^ From the
The RTG1591 is funded by the German Research Foundation (DFG). We are dedicated to training PhD students in research projects concerning the post-transcriptional control of gene expression in directing cell fate, human diseases and plant-pathogen interactions. Successful candidates will characterize, on the molecular level, how RNA-binding proteins, enzymes acting on RNA, and various regulatory RNAs, including microRNAs, long non-coding RNAs and bacterial sRNAs, modulate gene expression. On the basis of animal and plant model organisms, students will explore how the post-transcriptional control of gene expression determines cell fate and diseases. The RTG1591 provides a structured post-graduate training program including various workshops and seminars supporting students in establishing a successful scientific career. We offer MD stipends (stipend, approximately 830€/month) for one year with an optional extension for an additional two years.. ...
Tight junction-associated protein 1 is a protein that in humans is encoded by the TJAP1 gene. TJAP1 has been shown to interact with DLG1. GRCh38: Ensembl release 89: ENSG00000137221 - Ensembl, May 2017 GRCm38: Ensembl release 89: ENSMUSG00000012296 - Ensembl, May 2017 "Human PubMed Reference:". "Mouse PubMed Reference:". Kawabe H, Nakanishi H, Asada M, Fukuhara A, Morimoto K, Takeuchi M, Takai Y (Dec 2001). "Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells". J Biol Chem. 276 (51): 48350-5. doi:10.1074/jbc.M107335200. PMID 11602598. "Entrez Gene: TJAP1 tight junction associated protein 1 (peripheral)". Maruyama K, Sugano S (1994). "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides". Gene. 138 (1-2): 171-4. doi:10.1016/0378-1119(94)90802-8. PMID 8125298. Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, et al. (1997). "Construction and characterization of a full length-enriched and a 5-end-enriched cDNA library". ...
The ability to accurately repair DNA damaged by spontaneous errors, oxidation or mutagens is crucial to the survival of cells. This repair is normally accomplished by using an identical or homologous intact sequence of DNA, but scientists have now shown that RNA produced within cells of a common budding yeast can serve as a template for repairing the most devastating DNA damage - a break in both strands of a DNA helix.. Earlier research had shown that synthetic RNA oligonucleotides introduced into cells could help repair DNA breaks, but the new study is believed to be the first to show that a cells own RNA could be used for DNA recombination and repair. The finding provides a better understanding of how cells maintain genomic stability, and if the phenomenon extends to human cells, could potentially lead to new therapeutic or prevention strategies for genetic-based disease.. The research was supported by the National Science Foundation, the National Institutes of Health and the Georgia Research ...
0231] U.S. patents disclosing protease inhibitors for the treatment of HCV include, for example, U.S. Pat. No. 6,004,933 to Spruce et al., which discloses a class of cysteine protease inhibitors for inhibiting HCV endopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al., which discloses synthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No. 5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc., and U.S. Pat. No. 7,169,760, US2005/176648, WO 02/08187 and WO 02/008256 to Schering Corporation. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serine protease inhibitors of HCV are disclosed in WO 02/48172 and U.S. Pat. No. 6,911,428 to Schering Corporation. Imidazoleidinones as NS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 and U.S. Pat. No. 6,838,475 to Schering Corporation and WO 02/48157 and U.S. Pat. No. 6,727,366 to ...
DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted ...
Looking for online definition of phosphodiester bond in the Medical Dictionary? phosphodiester bond explanation free. What is phosphodiester bond? Meaning of phosphodiester bond medical term. What does phosphodiester bond mean?
The present invention provides novel polynucleotides encoding HGPRBMY23 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel HGPRBMY23 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly renal diseases and/or disorders, colon cancer, breast cancer, and diseases and disorders related to aberrant NFKB modulation. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.