Description: Two serious pathogens of mammals, Cryptococcus neoformans and Cryptococcus gattii , comprise the C. neoformans species complex in the fungal phylum Basidiomycota. These yeasts are facultative intracellular microbes: in the environment they grow either independently or possibly within soil amoeba (Steenbergen, et al., 2001), and in mammalian hosts they similarly occur either free in tissues or body fluids or within phagocytic cells. The importance of C. neoformans to human health has stimulated its development as an experimental model for both basic physiology and pathogenesis. This article is protected by copyright. All rights reserved.

Description: Whole genome sequencing has revealed large quantities of genetic information for many yeast species. These data include gene maps, gene content, and gene functions, providing an opportunity for investigating fungal species other than the model organism of baker's yeast, Saccharomyces cerevisiae . One such alternative fungal species is Schizosaccharomyces japonicus . This article is protected by copyright. All rights reserved.

Description: A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae , the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20 T treatment was higher than that from the wild-type strain. The results of GFP and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The level of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p -nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. This article is protected by copyright. All rights reserved.

Description: 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 generation of live cells lacking cell wall, called protoplasts, that can be maintained in osmostabilised 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 wall with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than ten minutes. We demonstrate that these protoplasts can be utilised 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. Highlights A novel method to prepare protoplasts from fission yeast is reported This method is rapid (10-20 minutes) and efficient (100% cells become protoplasts) The method is useful for diploid generation or plasmid extraction Lallzyme MMX is a good alternative to novozyme and zymolyase This article is protected by copyright. All rights reserved.

Description: The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR amplified separately, mixed and directly used for the transformation. URA3 + transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis and it was included in the primers to amplify target DNAs for cloning. Transformation with PCR amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among DNA cloning systems developed to date. This article is protected by copyright. All rights reserved.

Description: Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S . cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species. The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European, and Sake) and represent clean, non-mosaic, lineages of S . cerevisiae , meaning that their genomes differ essentially by monomorphic and private SNPs. In this work, one representative strain for each of the five S . cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions so that all dominant markers remain available for further genetic manipulations. A set of 50 strains was assembled, including 8 haploid and 2 diploid strains for each lineage. These strains carry different combinations of leu2Δ0 , lys2Δ0 , met15Δ0 , ura3Δ0 and/or ura3Δ::KanMX-barcoded deletions with marker configuration resembling that of the BY series which will allow large-scale crossing with existing deletion collections. This new set of genetically tractable strains provides a powerful toolkit to explore the impact of natural variation on complex biological processes. This article is protected by copyright. All rights reserved.

Description: The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting, or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One-step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3 . Here we describe the PCR-based Fifty-Fifty method for seamless genome editing that requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop-in/pop-out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the Fifty-Fifty method to make two conservative loss-of-function mutations in MATALPHA1 , with results that suggest the wildtype gene has a new function outside of that presently known. This article is protected by copyright. All rights reserved.

Description: Human interferon alpha-2b (hIFNα2b) is the most important member of interferon family. Escherichia coli , yeast, mammalian cell cultures and baculovirus infected insect cells have been used for expressing recombinant human interferon. Recently Pichia pastoris based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered, an important aspect to get the optimum expression of recombinant protein which may vary from one protein to another. In present study, we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris . Constructs containing from one to five repeats of IFNα2b expressing cassettes were created via in vitro multimerization approach. P. pastoris host strain X-33 was transformed with these expression cassettes. Groups of P. pastoris clones transformed with different copies of IFNα2b expression cassette were screened for intrachromosomal integration. IFNα2b expression level of stable transformants was checked. Copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that increase in copy number generally have positive effect on expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b . This was also observed that increase in drug resistance of a clone not guarantee its high expression, as integration of marker gene do not always correlate with integration of gene of interest. This article is protected by copyright. All rights reserved.

Description: Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high throughput methods of assessing yeast cell viability such as flow-cytometry and SGA analysis do not measure the percent viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called Tadpoling, to quantify the percent viability of a yeast culture with the ability to detect as few as one viable cell amongst ~10 8 dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish we are able to observe a range of viability from 100% to .0001%. Comparison of Tadpoling to the traditional plating method to measure yeast culture viability reveals that for the majority of Saccharomyces species analyzed, there is no significant difference between the two methods. In comparison to flow-cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, Tadpoling is much more accurate at culture viabilities below 1%. Thus, we show that Tadpoling provides an easy, inexpensive, space-saving method amenable to high-throughput screens for accurately measuring yeast cell viability. This article is protected by copyright. All rights reserved.

Description: When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K + ) had varied effects on the respiration-induced yPTP depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K + inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K + partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg 2+ , NH 4 + , or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. This article is protected by copyright. All rights reserved.