Application of run-on transcription method for studying the regulation of plastid genome expression

This paper describes in detail the main stages of the run-on transcription assay, which permits studying the regulation of the transcription rates for nuclear, chloroplast, and mitochondrial genes. This is very important for understanding the action mechanisms of phytohormones, light, temperature, and other factors. This method includes isolation of DNA-containing organelles, in vitro transcription in their lysates in the presence of labeled ³²P-precursors of RNA, isolation of newly synthesized transcripts, and their hybridization with gene-specific DNA fragments fixed on a special membrane. We discuss some possible difficulties in the assay performing and some occasionally met inaccuracies in the result interpretation. In spite of a great potential of this method, it is not essentially applied by Russian researchers, in particular those studying plant biology. The objective of this work was to facilitate a rapid introduction of the run-on transcription method in the practice of scientific research of Russian plant physiologists.