transcriptional regulation, chloroplast isolation, run-on transcription assay, Cyanidioschyzon merolae
Chloroplasts are plant-specific, semi-autonomous organelles that are derived from an endosymbiotic event involving ancestral cyanobacteria, oxygenevolving photosynthetic prokaryotes. Chloroplasts possess their own genome and gene expressin machineries that are of bacterial origin. However, most symbiont genes have been lost or transferred into the host nuclear genome. Thus, coordination of nuclear and chloroplast gene expression is essential to maintaining normal chloroplast functions including photosynthesis. To understand the transcriptional regulation of chloroplast genes in a wide range of plants, several biochemical methods such as run-on transcription assays and in vitro transcription analyses have been employed. These require the preparation of isolated intact chloroplasts. In this paper, we describe a convenient protocol for the isolation of intact chloroplasts from a red alga, Cyanidioschyzon merolae, as well as a run-on transcription assay, for the subsequent analysis of isolated chloroplasts.