A New Method for the Preparation of Highly Purified and More Native Nuclei Showing Stage-Specific Transcription of Actin and Tubulin Genes

Abstract

Physarum nuclei usually have been isolated by the standard procedure of Mohberg and Rusch (1971). This Triton-sucrose method (TS method) yields nuclei containing up to seven times more polysaccharides (mainly slime) than DNA (Hall et al., 1975). Some have presumed that the slime is located inside the nucleus (Farr and Horrisberger, 1978); it has been shown to have deleterious effects on enzymatic treatments of chromatin (Staron et al., 1980). Therefore, we have developed a new method of nuclear isolation. It was recently demonstrated that Triton X-100 dissolves nuclear membranes (Frederiks et al., 1978). We chose to use Surfynol as detergent because it keeps the nuclear membranes widely intact (Fig. 19-1). In order to reduce leakage during the isolation procedure, we used hexylene glycol instead of sucrose and, for higher purification, a Percoll density gradient instead of a sucrose cushion. For this reason, the new method is called the SHP-method (Nothacker and Hildebrandt, 1985, 1986). Percoll density gradients were also used earlier for the purification of nucleoli from contaminating slime (Seebeck et al., 1979). Recently, nuclei were prepared by being pelleted through a cushion of Percoll (28% v/v) instead of 1 M sucrose (Künzler et al., 1984).