Isolation of a double‐stranded RNA and a virus‐like particle from Geotrichum candidum

Abstract

A virus-like particle (VLP) of 42 nm diameter was isolated from the lipolytic microorganism Geotrichum candidum. The VLP contains a linear double-stranded RNA molecule of 1.70 pm in length. Proteins of VLP were studied by polyacryl amide gel electrophoresis.A lot of fungi contain virus-like particles (VLPs), the genome of which consists of double-stranded RNA (ds RNA) molecules. These VLPs have been found in Penicillium: Penicillium. stoloniferum (KLEINSCHMIDT et In this work, we report the isolation of some of these particles from Geotrichum candidum CBS 17853 strains, and give characteristics of their acid nucleic components. Materials and methodsBiological material: The Geotrichum candidum LINK CBS 17853, 18767, 11523 strains were used. Cultures were performed in liquid YPG medium (1 % w/v yeast extract DIFCO, I % w/v bacto-peptone DIFCO, 2 v% w/v glucose PROLABO). Aerobic cultures were grown, using a reciprocating shaker (80 oscillations/min, amplitude of 7 cm) and temperature was regulated at 28 "C. Cells were harvested at the end of the log phase.Extraction of nucleic acids: Nucleic acids were extracted from cells by the method of HOLM et al. (1986). These nucleic acids were preserved at -20 "C after precipitation by cold ethanol.Purification of viral RNA: The purification was carried from total nucleic acids (MATTE et al. 1990). The total material was layered on sucrose gradient 15-40% after heating to 68°C for 10min to improve the separation of different nucleic acids. Then an ultracentrifugation was carried on a swing rotor (BECKMAN, SW41Ti) at speed 25000 rpm for 20 h and 25 "C. The band of viral RNA so purified was punctered, dried and redissolved into specific buffer following use.Agarose gel electrophoresis: Following the method of MANIATIS et al. (1982), the precipitated nucleic acids were resuspended in loading buffer (TE buffer: Tris-HC1 10 mM; EDTA 1 mM; pH 7.5 and bromophenol blue 0.15 mg.ml-'. Electrophoresis was carried out in 1% w/v agarose gel containing 1 pg.rn1-l ethidium bromide run for 2 h at 80 V in TAE buffer (Tris-HC1 40 mM; sodium acetate 20 mM; EDTA 1 mM, pH 8.0). Gels were visualized and photographed on a short UV transilluminator (VILBERT-LOURMAT) using a Polaroid CU-5 land camera.Study of RNA: Enzymatic tests: RNA samples were digested with the following enzymes, according to the experiments: DNase: The solution of nucleic acid (50 pg.ml-') was incubated in buffer sodium

Molecular Characterization of Two Totiviruses from the Commensal Yeast Geotrichum candidum

“…Yeast fungi have been long known as hosts of mycoviruses since the discovery of a killer phenotype associated with Saccharomyces-virus killer systems [34]. In G. candidum, uncharacterized dsRNAs and virus-like particles have been discovered in the past [21,22]. Recently, three species belonging to the family Totiviridae have been characterized at the molecular level [23].…”

“…The known host range of Totiviridae members and totivirus-like genomes has expanded over recent years, with the increased utilization of next-generation sequencing technologies, to include insects, arthropods, fish, crab, and plants [16][17][18][19][20]. Historically, virus-like particles (VLPs) and dsRNAs have been detected in G. candidum without further biological or molecular identification of these associated VLPs or dsRNAs [21,22]. Recently, four totiviruses were detected and characterized from G. candidum isolates from Pakistan [23].…”

Isolation and characterization of the dsRNA virus from the yeast Endomyces magnusii

“…Many species of fungi contain non-infectious mycoviruses. Virus-like particles (VLPs) have been found in the yeasts Saccharomyces cerevisiae (for reviews see [1,2]), Yarrowia lipolytica [3] Candida tropicalis [4,5], Candida albicans [6] and in the yeast-like microorganism Geotrichum candidum [7]. The genetic material of mycoviruses consists of dsRNA segments of unequal length which are separately encapsulated in the VLPs.…”