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2004
Grosjean H., Keith G., Droogmans L.
Detection and quantification of modified nucleotides in RNA using thin-layer chromatography Book Section
Dans: Gott, J. M. (Ed.): RNA Interference, Editing, and Modification: Methods and Protocols, vol. 265, p. 357-91, Springer Protocols, Humana Press, 2004.
Résumé | BibTeX | Étiquettes: &, 5', acids, and, Base, Chromatography, Composition, Deoxyribonucleotides/chemistry/isolation, DNA/chemistry/genetics/isolation, Endoribonucleases, Gov't, Indicators, Isotope, KEITH, Labeling/methods, Layer/methods, Non-U.S., Nucleic, Oligoribonucleotides/chemistry/isolation, Peptide, purification, Radioisotopes, Reagents, Regions/chemistry, Ribonucleotides/*analysis, RNA/*genetics/isolation, Support, Thin, Untranslated
@incollection{,
title = {Detection and quantification of modified nucleotides in RNA using thin-layer chromatography},
author = { H. Grosjean and G. Keith and L. Droogmans},
editor = { J.M. Gott},
year = {2004},
date = {2004-01-01},
booktitle = {RNA Interference, Editing, and Modification: Methods and Protocols},
volume = {265},
pages = {357-91},
publisher = {Springer Protocols, Humana Press},
series = {Methods in Molecular Biology},
abstract = {Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50-150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and two-dimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.},
keywords = {&, 5', acids, and, Base, Chromatography, Composition, Deoxyribonucleotides/chemistry/isolation, DNA/chemistry/genetics/isolation, Endoribonucleases, Gov't, Indicators, Isotope, KEITH, Labeling/methods, Layer/methods, Non-U.S., Nucleic, Oligoribonucleotides/chemistry/isolation, Peptide, purification, Radioisotopes, Reagents, Regions/chemistry, Ribonucleotides/*analysis, RNA/*genetics/isolation, Support, Thin, Untranslated},
pubstate = {published},
tppubtype = {incollection}
}
Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50-150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and two-dimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.
1993
Pfeiffer P., Jung J. L., Heitzler J., Keith G.
Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba Article de journal
Dans: J Gen Virol, vol. 74, non Pt 6, p. 1167-73, 1993, (0022-1317 Journal Article).
Résumé | BibTeX | Étiquettes: *Plants, &, Base, Bodies, Cytoplasm, Data, Extrachromosomal, Fabaceae/*genetics, Genetic, Inclusion, Infertility/genetics, Inheritance/*genetics, Medicinal, Molecular, Pollen/genetics, purification, Replicase/metabolism, RNA, RNA/*genetics/isolation, Sequence, Transcription, Viral
@article{,
title = {Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba},
author = { P. Pfeiffer and J. L. Jung and J. Heitzler and G. Keith},
year = {1993},
date = {1993-01-01},
journal = {J Gen Virol},
volume = {74},
number = {Pt 6},
pages = {1167-73},
abstract = {The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [alpha-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait.},
note = {0022-1317
Journal Article},
keywords = {*Plants, &, Base, Bodies, Cytoplasm, Data, Extrachromosomal, Fabaceae/*genetics, Genetic, Inclusion, Infertility/genetics, Inheritance/*genetics, Medicinal, Molecular, Pollen/genetics, purification, Replicase/metabolism, RNA, RNA/*genetics/isolation, Sequence, Transcription, Viral},
pubstate = {published},
tppubtype = {article}
}
The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [alpha-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait.
