Measurement of Transcription Rates in Arabidopsis Chloroplasts
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.
Key wordsArabidopsis thaliana Chloroplast Gene expression Run-on assay Transcription rate Plastid isolation DNA/RNA hybridization
This work was funded by Deutsche Forschungsgemeinschaft (SFB 429 to T.B. and K/L.). We thank Liliana Borsellino, Dr. Victor V. Kusnetsov, and Dr. Maria V. Yamburenko for their helpful discussions.
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