title: A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.
abstract: In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.
authors.affiliation: Unit for Molecular Genetics, Karolinska Institute, Huddinge, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Nyholm
authors.forename: M
authors.initials: M
authors.lastname: Andäng
authors.forename: G
authors.initials: G
authors.lastname: Hotchkiss
authors.forename: T
authors.initials: T
authors.lastname: Härd
authors.forename: H
authors.initials: H
authors.lastname: Baumann
authors.forename: S
authors.initials: S
authors.lastname: Larsson
authors.forename: L
authors.initials: L
authors.lastname: Ahrlund-Richter
mesh_terms: D002247:Carbon Isotopes; D002701:Chloramphenicol / Q000494:pharmacology; D017046:Cost Savings; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D009587:Nitrogen Isotopes; D010710:Phosphates / Q000737:chemistry* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000187:drug effects; D012265:Ribonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010710:Phosphates; D012329:RNA, Bacterial; D012265:Ribonucleotides; D002701:Chloramphenicol