Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats.

Abstract:

:We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and 29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Osborne RJ,Thornton CA

doi

10.1093/nar/gkn025

subject

Has Abstract

pub_date

2008-03-01 00:00:00

pages

e24

issue

4

eissn

0305-1048

issn

1362-4962

pii

gkn025

journal_volume

36

pub_type

杂志文章
  • Nicking is asynchronous and stimulated by synapsis in 12/23 rule-regulated V(D)J cleavage.

    abstract::The first step in DNA cleavage at V(D)J recombination signals by RAG1 and RAG2 is creation of a nick at the heptamer/coding flank border. Under proper conditions in vitro the second step, hairpin formation, requires two signals with spacers of 12 and 23 bp, a restriction referred to as the 12/23 rule. Under these cond...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/25.21.4370

    authors: Eastman QM,Schatz DG

    更新日期:1997-11-01 00:00:00

  • Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination.

    abstract::We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3' ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR tha...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkw275

    authors: Onyango DO,Howard SM,Neherin K,Yanez DA,Stark JM

    更新日期:2016-07-08 00:00:00

  • Molecular characterization of the MT-family of dispersed middle-repetitive DNA in rodent genomes.

    abstract::We report the consensus sequence of members of a new dispersed middle-repetitive DNA family, MT, which is present in mouse and rat genomes. This family is shown to be as abundant as the described rodent B1 and B2 families. Hybridization experiments with radioactive single-stranded cDNAs from different tissues indicate...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/14.16.6403

    authors: Heinlein UA,Lange-Sablitzky R,Schaal H,Wille W

    更新日期:1986-08-26 00:00:00

  • Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

    abstract::Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/11.5.1213

    authors: Chebath J,Merlin G,Metz R,Benech P,Revel M

    更新日期:1983-03-11 00:00:00

  • Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta.

    abstract::Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfa...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/29.22.4607

    authors: Boudsocq F,Iwai S,Hanaoka F,Woodgate R

    更新日期:2001-11-15 00:00:00

  • MalaCards: an amalgamated human disease compendium with diverse clinical and genetic annotation and structured search.

    abstract::The MalaCards human disease database (http://www.malacards.org/) is an integrated compendium of annotated diseases mined from 68 data sources. MalaCards has a web card for each of ∼20 000 disease entries, in six global categories. It portrays a broad array of annotation topics in 15 sections, including Summaries, Symp...

    journal_title:Nucleic acids research

    pub_type: 杂志文章,评审

    doi:10.1093/nar/gkw1012

    authors: Rappaport N,Twik M,Plaschkes I,Nudel R,Iny Stein T,Levitt J,Gershoni M,Morrey CP,Safran M,Lancet D

    更新日期:2017-01-04 00:00:00

  • An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation.

    abstract::During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pat...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkw172

    authors: Toribio R,Díaz-López I,Boskovic J,Ventoso I

    更新日期:2016-05-19 00:00:00

  • NXSensor web tool for evaluating DNA for nucleosome exclusion sequences and accessibility to binding factors.

    abstract::Nucleosomes, a basic structural unit of eukaryotic chromatin, play a significant role in regulating gene expression. We have developed a web tool based on DNA sequences known from empirical and theoretical studies to influence DNA bending and flexibility, and to exclude nucleosomes. NXSensor (available at http://www.s...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkl158

    authors: Luykx P,Bajić IV,Khuri S

    更新日期:2006-07-01 00:00:00

  • Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

    abstract::T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate ca...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/30.4.1073

    authors: Wang LK,Shuman S

    更新日期:2002-02-15 00:00:00

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

    abstract::Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. Th...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/14.18.7305

    authors: Vishwanatha JK,Yamaguchi M,DePamphilis ML,Baril EF

    更新日期:1986-09-25 00:00:00

  • The conserved 3'-flanking sequence, AATGGAAATG, of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA.

    abstract::We examined the 3'-flanking regions required for accurate 3'-end formation of wheat histone H3 mRNA using gene expression in transformed sunflower cells. The introduction of mutations into the conserved sequence AATGGAAATG in the 3'-flanking region of plant histone genes, located 22 bp upstream from the polyadenylatio...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/22.6.1052

    authors: Ohtsubo N,Iwabuchi M

    更新日期:1994-03-25 00:00:00

  • Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor.

    abstract::Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, t...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkw095

    authors: Morales-Hernández A,González-Rico FJ,Román AC,Rico-Leo E,Alvarez-Barrientos A,Sánchez L,Macia Á,Heras SR,García-Pérez JL,Merino JM,Fernández-Salguero PM

    更新日期:2016-06-02 00:00:00

  • Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

    abstract::The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, su...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/23.6.885

    authors: Bailly C,Waring MJ

    更新日期:1995-03-25 00:00:00

  • Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates.

    abstract::The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 speci...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkf486

    authors: Fujimoto K,Iida H,Kawakami M,Bando T,Tao ZF,Sugiyama H

    更新日期:2002-09-01 00:00:00

  • Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe.

    abstract::The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosacc...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkf334

    authors: Chakraborty S,Sarmah B,Chakraborty N,Datta A

    更新日期:2002-07-01 00:00:00

  • Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds.

    abstract::We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/16.9.3671

    authors: Chu BC,Orgel LE

    更新日期:1988-05-11 00:00:00

  • Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin.

    abstract::The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF st...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkf479

    authors: De Silva IU,McHugh PJ,Clingen PH,Hartley JA

    更新日期:2002-09-01 00:00:00

  • A nascent micronuclear pseudogene in the ciliate Euplotes crassus.

    abstract::The macronuclear genome of Euplotes crassus contains two different genes, EFA1 and EFA2, encoding EF-1alpha proteins. They are derived from micronuclear precursors in the course of a sexual process termed conjugation. We have found that two apparent micronuclear precursors exist for EFA1. They differ in their potentia...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/24.16.3195

    authors: Florian V,Klein A

    更新日期:1996-08-15 00:00:00

  • The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes.

    abstract::We have isolated the gene coding for the U2 analogue in trypanosomes. The 148 nucleotide long U2 RNA is capped and transcribed from a single copy gene. The 5' half of the molecule is highly homologous to mammalian U2 RNA, while the 3' half does not show any significant sequence homology with the mammalian counterpart....

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/14.22.8893

    authors: Tschudi C,Richards FF,Ullu E

    更新日期:1986-11-25 00:00:00

  • Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants.

    abstract::The cDNA encoding the human RNA lariat debranching enzyme (hDBR1) was identified and cloned by searching the Expressed Sequence Tag (EST) database and screening a HeLa cDNA library, based on predicted amino acid sequence homologies with the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Caenorhabditis elegans...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/28.18.3666

    authors: Kim JW,Kim HC,Kim GM,Yang JM,Boeke JD,Nam K

    更新日期:2000-09-15 00:00:00

  • SEASTAR: systematic evaluation of alternative transcription start sites in RNA.

    abstract::Alternative first exons diversify the transcriptomes of eukaryotes by producing variants of the 5' Untranslated Regions (5'UTRs) and N-terminal coding sequences. Accurate transcriptome-wide detection of alternative first exons typically requires specialized experimental approaches that are designed to identify the 5' ...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gky053

    authors: Qin Z,Stoilov P,Zhang X,Xing Y

    更新日期:2018-05-04 00:00:00

  • Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins.

    abstract::DNA replication of phi29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi29-like DNA replication. In the present work we analyze th...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/28.10.2034

    authors: Gascón I,Lázaro JM,Salas M

    更新日期:2000-05-15 00:00:00

  • SNPWave: a flexible multiplexed SNP genotyping technology.

    abstract::Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gnh045

    authors: van Eijk MJ,Broekhof JL,van der Poel HJ,Hogers RC,Schneiders H,Kamerbeek J,Verstege E,van Aart JW,Geerlings H,Buntjer JB,van Oeveren AJ,Vos P

    更新日期:2004-03-05 00:00:00

  • Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme.

    abstract::Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presen...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkh753

    authors: Inoue A,Takagi Y,Taira K

    更新日期:2004-08-09 00:00:00

  • Polylysine-coated mica can be used to observe systematic changes in the supercoiled DNA conformation by scanning force microscopy in solution.

    abstract::The conformations of supercoiled (sc) DNA and linear DNA bound to polylysine (PL)-coated mica were investigated by scanning force microscopy (SFM) in solution. From the polymer statistical analysis of linear DNA, we could distinguish between re-arrangements or trapping of the DNA on the surface. Conditions of re-arran...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gng137

    authors: Bussiek M,Mücke N,Langowski J

    更新日期:2003-11-15 00:00:00

  • Affinity proteomic dissection of the human nuclear cap-binding complex interactome.

    abstract::A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkaa743

    authors: Dou Y,Kalmykova S,Pashkova M,Oghbaie M,Jiang H,Molloy KR,Chait BT,Rout MP,Fenyö D,Jensen TH,Altukhov I,LaCava J

    更新日期:2020-10-09 00:00:00

  • Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification.

    abstract::In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gku249

    authors: Neumann P,Lakomek K,Naumann PT,Erwin WM,Lauhon CT,Ficner R

    更新日期:2014-06-01 00:00:00

  • PlantLoc: an accurate web server for predicting plant protein subcellular localization by substantiality motif.

    abstract::Knowledge of subcellular localizations (SCLs) of plant proteins relates to their functions and aids in understanding the regulation of biological processes at the cellular level. We present PlantLoc, a highly accurate and fast webserver for predicting the multi-label SCLs of plant proteins. The PlantLoc server has two...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkt428

    authors: Tang S,Li T,Cong P,Xiong W,Wang Z,Sun J

    更新日期:2013-07-01 00:00:00

  • Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants.

    abstract::The petunia rbcS gene SSU301 was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. The time at which rbcS expression was maximal after transfer of the tobacco plants to the greenhouse was determined. The expression level of the SSU301 gene varied up to 9 fold between individual tobacco p...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/16.19.9267

    authors: Dean C,Jones J,Favreau M,Dunsmuir P,Bedbrook J

    更新日期:1988-10-11 00:00:00

  • Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.

    abstract::Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) ...

    journal_title:Nucleic acids research

    pub_type: 杂志文章

    doi:10.1093/nar/gkt1321

    authors: Kumar P,Sweeney TR,Skabkin MA,Skabkina OV,Hellen CU,Pestova TV

    更新日期:2014-03-01 00:00:00