A Lallzyme MMX‐based rapid method for fission yeast protoplast preparation

Abstract

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi.

Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe

“…Nuclear spreads of meiotic Sz. pombe cells were prepared as described previously (Loidl and Lorenz 2009 ), with the only modification that Lallzyme MMX (Lallemand Inc., Montréal, Canada) at a final concentration of 100 mg/ml was used as the sole enzyme in the spheroplasting solution (Flor-Parra et al 2014 ).…”

Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy

“…Single diploid colonies were counted on day 7. Alternatively, h − /h − diploids were constructed by protoplast fusion 38 and isolated in PMG –ADE +sorbitol and subsequently treated as described above. For the LOH assay, diploid colonies were replica plated onto YEA +NAT for scoring the NAT S fraction, and onto EMM medium at 30°C to induce sporulation.…”

Intragenic meiotic recombination in Schizosaccharomyces pombe is sensitive to environmental temperature changes

“…Meiotic time-courses and preparation of chromatin spreads were in essence performed as published (Loidl and Lorenz 2009), except for the use of 100 mg/ml Lallzyme MMX (Lallemand Inc., Montréal, Canada) as the cell-wall digesting enzyme (Flor-Parra et al 2014). Immunostaining was performed according to an established protocol (Loidl and Lorenz 2009) using polyclonal rabbit α-myc (ab9106; Abcam PLC, Cambridge, UK) at a 1:500 dilution and monoclonal rat α-GFP [3H9] (ChromoTek GmbH, Planegg-Martinsried, Germany) at a 1:100 dilution as primary antibodies.…”