Isolation of plant nuclei.

The isolation of plant nuclei is a useful first step in many experiments concerned with the mechanism and control of gene expression in plants. For example, isolated nuclei can be used for the isolation of nuclear components such as chromosomal proteins (1), for the study of the processing of primary transcripts (2,3), for the assay and characterization of RNA polymerase activities (4-7), or for the measurement of transcription rates of specific genes (8) (see Chapter 37 ). A method is described here for the isolation of a crude preparation of intact plant nuclei, with an additional protocol for nuclei purification on a discontinuous gradient of Percoll (9) (see Note 1 in section 4). Centrifugation of crude nuclei preparations through Percoll gradients removes much of the contaminating cytoplasmic material such as starch grains (10), and the Percoll step appears to reduce the ribonuclease activity associated with nuclei (10). It is therefore recommended for transcription experiments. The crude nuclei preparation alone may be adequate for some work, for example, when attempting RNA polymerase assays for the first time, when very small amounts of tissue are involved, for preliminary experiments on the characterization of enzyme activities, or for the isolation of nuclear components that may be lost during purification.