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 Neurospora 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 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 ,um in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride 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 molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride

“…We obtained a reproducible yield of about 0.3-0.4 mg DNA/g wet wt by HF treatment of C. graeile. This value is comparable to that obtained using French press treatment [11]. The recovery of S. cerevisiae DNA after treatment with HF was 4/zg DNA/3 × 108 cells or approx.…”

Light-regulated protein and poly(A)+ mRNA synthesis in Neurospora crassa.

“…In line with others, we found that poly(A)+ RNA constituted 1.5 % of total RNA (Hautala et al, 1977;Lucas et al, 1977). This RNA was capable of stimulating protein synthesis in the rabbit reticulocyte lysate system by 40-fold or greater.…”

Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor

“…The extraction medium contained 1.0 M sorbitol, 5% Ficoll, 20% glycerol, 5 mM MgCl2, 10 mM CaCl2, and 0.5% Triton X-100, a minor modification of buffer A of Hautala et al (10). The glass beads were washed once with the same medium to give a combined volume of original extract and wash of 7 to 8 ml/g of mycelia.…”

“…Preparation of cell fractions. Crude nuclear pellets were prepared from the extract by the method of Hautala et al (10), resuspended in 2.5 ml of the detergent-containing extraction medium per g of mycelia (see above), and filtered through glass wool to remove pieces of unbroken mycelia. The nuclei were then resedimented and resuspended in 1 ml of buffer B of Hautala et al (10), which does not contain Triton X-100, per g of mycelia.…”