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 nuclear extracts. This is especially true for etiolated seedlings. Here we repor...

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Bibliographic Details
Published in:Plant cell reports 2000-04, Vol.19 (5), p.504-510
Main Authors: FOLTA, K. M, KAUFMAN, L. S
Format: Article
Language:English
Subjects:
Arabidopsis thaliana
Biological and medical sciences
Biotechnology
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
Others
Seedlings
Various methods and equipments
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Summary: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 nuclear extracts. This is especially true for etiolated seedlings. Here we report conditions, optimized for use in Arabidopsis, which allow for the isolation of enriched fractions of functional nuclei from less than 2 g of etiolated or light-grown tissue. The nuclei are capable of incorporating H-UTP into TCA-insoluble RNA, and also incorporate P-CTP into transcripts that can subsequently be hybridized to specific filter-bound DNA target sequences. The functional nuclei are sensitive to the transcriptional inhibitors actinomycin D and α-amanitin, confirming that the transcription observed is both template dependent and relies on nuclear polymerase II.
ISSN:0721-7714
1432-203X
DOI:10.1007/s002990050764