title: Measurement of transcription rates in Arabidopsis chloroplasts.
abstract: The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a protocol for run-on transcription in chloroplasts isolated from Arabidopsis leaves and present data on the transcriptional activity of several plastid genes in detached leaves of different Arabidopsis ecotypes.
authors.affiliation: Institut für Biologie (Genetik), Humboldt-Universität zu Berlin, Berlin, Germany.
authors.forename: Yan O
authors.initials: YO
authors.lastname: Zubo
authors.forename: Thomas
authors.initials: T
authors.lastname: Börner
authors.forename: Karsten
authors.initials: K
authors.lastname: Liere
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D061125:Genes, Chloroplast / Q000235:genetics; D005821:Genetic Techniques; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D018747:RNA, Chloroplast / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D008567:Membranes, Artificial; D018747:RNA, Chloroplast