Isolation and characterization of nuclei from Neurospora crassa

Abstract

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of N. crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20-30%. The nuclei had a DNA:RNA:protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 .mu.m in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70-75% yield by dissociation with 2 M NaCl and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in MW from 15,000-70,000. The gel pattern was more complex for total dissociated chromatin than for reconstituted chromatin.