Improving poor in vitro transcription from G,C-rich genes

Aziz, Ruksana; Soreq, Hermona

doi:10.1093/nar/18.11.3418

Cited by 12 publications

(1 citation statement)

References 4 publications

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“…In vitro transcription and RNA transfections. RNA transcripts were synthesized in vitro with T7 RNA polymerase, under conditions adapted from previously published methods (3,26,55), from a template consisting of the full-length cDNA of the BVDV genome contained in pVVNADL linearized with SacII and treated with T4 to remove the 3Ј overhang. Capped RNA was synthesized with a T7 RNA polymerase in vitro transcription kit (Ambion, part 1344).…”

Section: Methodsmentioning

confidence: 99%

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts

Vassilev

Collett

Donis

1997

J Virol

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Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5 and 3 termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5 untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.

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Section: Methodsmentioning

confidence: 99%

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts

Vassilev

Collett

Donis

1997

J Virol

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Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase

Gruegelsiepe

Schön

Kirsebom

et al. 2014

Handbook of RNA Biochemistry

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Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Neville¹,

Gnatt²,

Loewenstein³

et al. 1992

The EMBO Journal

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Structure‐function relationships of cholinesterases (CHEs) were studied by expressing site‐directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site‐directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of ‘atypical’ BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co‐appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N′ and C′ terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build‐up of the CHE active center.

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Improving poor in vitro transcription from G,C-rich genes

Cited by 12 publications

References 4 publications

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts

Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

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