Preparation of transcriptionally active nuclei from etiolated Arabidopsis thaliana

Despite its emergence as the plant model system, there are few reports that describe protocols for the isolation of functional nuclei from Arabidopsis thaliana and their use in nuclear run-on assays or in preparation of nucle ar extracts. This is especially true for etiolated seedlings. Here we repor t conditions, optimized for use in Arabidopsis, which allow for the isolati on of enriched fractions of functional nuclei from less than 2 g of etiolat ed or light-grown tissue. The nuclei are capable of incorporating H-3-UTP i nto TCA-insoluble RNA, and also incorporate P-32-CTP into transcripts that can subsequently be hybridized to specific filter-bound DNA target sequence s. The functional nuclei are sensitive to the transcriptional inhibitors ac tinomycin D and alpha-amanitin, confirming that the transcription observed is both template dependent and relies on nuclear polymerase II.