Isolation of nuclei from Aspergillus nidulans protoplasts

Abstract

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological state, as demonstrated by fluorescence microscopy and electronmicroscopy. The weight ratio of DNA:RNA:protein was 1:3.0:10.8 in the nuclear fraction.

Lovastatin possesses a fungistatic effect againstCandida albicans, but does not trigger apoptosis in this opportunistic human pathogen

“…For membrane fluidity measurements, protoplasts (Vágvölgyi & Ferenczy, 1991) from lovastatin-treated (5 mg mL À1 ) and untreated 15-h cultures were used. Sample preparations for spin labelling and electron paramagnetic resonance (EPR) measurements were carried out as described earlier (Belágyi et al, 1999;Pesti et al, 2000).…”

High-dose methylprednisolone influences the physiology and virulence ofCandida albicansambiguously and enhances the candidacidal activity of the polyene antibiotic amphotericin B and the superoxide-generating agent menadione

“…For membrane fluidity measurements, C. albicans protoplasts (Vágvölgyi & Ferenczy, 1991;Gyetvai et al, 2006) prepared from methylprednisolone-treated (4 mM), lovastatin-treated (5 mg mL À1 ) and untreated control 15-h cultures were used. Sample preparations for spin labeling and electron paramagnetic resonance (EPR) measurements with the fatty acid spin probe 5-doxylstearic acid were carried out as described elsewhere (Belágyi et al, 1999;Pesti et al, 2000;Gyetvai et al, 2006).…”

A simple method for isolation of nuclei from Basidiobolus ranarum (Zygomycota)

“…In the case of Aspergillus nidulans, on the other hand, a wildtype line was used (Gealt et al 1976), and the purity of nuclei was improved by density gradient centrifugation using Nycodentz (Vagvolgyi and Ferenczy 1991). Thus, most of the established methods for preparing fungal nuclei depend on cell lines, or ultracentrifugation with an expensive polydispersive agent.…”

“…In fungi and plants, the fi rst step of cell fraction involves maceration of the cell walls. Principally, this can be achieved by either mechanical disruption (Gealt et al 1976), enzymatic maceration (Vagvolgyi and Ferenczy 1991), or use of a wall-less mutant Received: November 19, 2008/ Accepted: May 22, 2009 Abstract A simple method for isolating the nuclei from Basidiobolus ranarum was established. To improve the yield and purity of nuclei, we investigated maceration methods, buffer composition, and centrifugation conditions to establish an optimal procedure.…”