Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method

Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially useful for laboratories using one or two strains repeatedly. The production of yeast cells for freezing takes only approximately 30 min, once the yeast culture has grown up. Transformation with frozen competent yeast cells will take approximately 30 min, depending on the heat shock used.     

Extraction of genomic DNA from yeasts for PCR-based applications

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

Marine yeasts as biocontrol agents and producers of bio-products

As some species of marine yeasts can colonize intestine of marine animals, they can be used as probiotics. It has been reported that β-glucans from marine yeast cells can be utilized as immuno-stimulants in marine animals. Some siderophores or killer toxins produced by marine yeasts have ability to inhibit growth of pathogenic bacteria or kill pathogenic yeasts in marine animals. The virulent factors from marine pathogens can be genetically displayed on marine yeast cells, and the yeast cells displaying the virulent factors can stimulate marine animals to produce specific antibody against the pathogens. Some marine yeast cells are rich in proteins and essential amino acids and can be used in nutrition for marine animals. The marine yeast cells rich in lipid can be used for biodiesel production. Recently, it has been reported that some strains of Yarrowia lipolytica isolated from marine environments can produce nanoparticles. Because many marine yeasts can remove organic pollutants and heavy metals, they can be applied to remediation of marine environments. It has been shown that the enzymes produced by some marine yeasts have many unique properties and many potential applications.     

Transformation of yeast by infectious prion particles

We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [PSI(+)] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.     

Encapsulation in a natural, preformed, multi-component and complex capsule: yeast cells

From the first observation about 40 years ago that yeast cells were interesting protective structures that could be used in several industrial applications, processes have been developed enabling technologists to incorporate several compounds possessing different physico-chemical (hydrophobic/hydrophilic) properties. Technologists screened yeast diversity to choose strains possessing the best potential and modified their physiological state to increase the uptake capability and the envelope plasticity, for instance by increasing the amount of lipids. Physico-chemical treatments were also used to improve the uptake and decrease the yeast natural material impact on the final products. For example, yeast cells could be “emptied” of their plasmic material. Yeast cells can also be coated with an additional polymeric material to increase resistance to heat treatment or decrease material liberation. These capsules can be used for several applications including carbonless paper, perfuming tissues and drug targeting, but the main industrial application deals currently with flavour encapsulation, although encapsulation in yeast is also interesting for the global food industry trend for health products. This paper proposes to review the use of yeast as an encapsulation structure focusing particularly on the properties of the yeast capsule and their impact on loading, protection, targeting and release.     

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.     

A microarray-based protocol for monitoring the growth of yeast overexpression strains

Gene overexpression can be used to investigate the biological pathways that are important in the response to a small molecule or other perturbation. To facilitate the use of gene overexpression in the study of small-molecule mechanisms, we developed a microarray-based protocol for monitoring the growth of a pool of yeast strains, each overexpressing a different protein. In this protocol, yeast harboring a set of approximately 3,900 galactose-inducible overexpression plasmids are grown in the absence or presence of a small molecule for multiple generations. The plasmids are then extracted from the two populations, processed and labeled in such a manner that their relative concentrations can be determined by competitive hybridization to a microarray. Although this protocol was developed for monitoring a specific set of overexpression plasmids, it could presumably be adapted to monitor yeast that have been transformed with any set of plasmids for which the gene inserts have been spotted, or otherwise arrayed, in a microarray format. This protocol can be completed in approximately 15 hours of hands-on time over the course of several days.     

Yarrowia lipolytica and pollutants: Interactions and applications

Yarrowia lipolytica is a dimorphic, non-pathogenic, ascomycetous yeast species with distinctive physiological features and biochemical characteristics that are significant in environment-related matters. Strains naturally present in soils, sea water, sediments and waste waters have inherent abilities to degrade hydrocarbons such as alkanes (short and medium chain) and aromatic compounds (biphenyl and dibenzofuran). With the application of slow release fertilizers, design of immobilization techniques and development of microbial consortia, scale-up studies and in situ applications have been possible. In general, hydrocarbon uptake in this yeast is mediated by attachment to large droplets (via hydrophobic cell surfaces) or is aided by surfactants and emulsifiers. Subsequently, the internalized hydrocarbons are degraded by relevant enzymes innately present in the yeast. Some wild-type or recombinant strains also detoxify nitroaromatic (2,4,6-trinitrotoluene), halogenated (chlorinated and brominated hydrocarbons) and organophosphate (methyl parathion) compounds. The yeast can tolerate some metals and detoxify them via different biomolecules. The biomass (unmodified, in combination with sludge, magnetically-modified and in the biofilm form) has been employed in the biosorption of hexavalent chromium ions from aqueous solutions. Yeast cells have also been applied in protocols related to nanoparticle synthesis. The treatment of oily and solid wastes with this yeast reduces chemical oxygen demand or value-added products (single cell oil, single cell protein, surfactants, organic acids and polyalcohols) are obtained. On account of all these features, the microorganism has established a place for itself and is of considerable value in environment-related applications.     

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein^1-3. Like many ''difficult'' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed ^4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described^6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)¹⁰. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.     

Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

Here, we describe a Library screen transformation protocol using the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method is suitable for screening complex plasmid libraries such as those used for yeast two-hybrid analysis. This procedure takes up to 2.5 h to complete once the yeast culture has been grown.     

Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols. This method has been used to purify YACs up to 600 kb in size. The purified YAC DNA is suitable for restriction enzyme digestion, DNA sequencing and functional studies. For example, YACs carrying full-size genes can be purified from yeast and used for transfection into mammalian cells or for the construction of a synthetic genome that can be used to produce a synthetic cell. This method for isolating high-quality YAC DNA in microgram quantities should be valuable for functional and synthetic genomic studies. The entire protocol takes ～3 d to complete.     

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.     

Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.     

一种简便高效的酵母基因组提取方法

提取基因组进行检测是酵母研究过程中的必要步骤之一。以毕赤酵母菌株GS115作为研究对象,主要成分为0.2 mol/L醋酸锂和1% SDS的酵母裂解液能高效的裂解酵母细胞壁。与两种酵母基因组提取试剂盒相比,该方法从相同体积的酵母培养液中获得的基因组的量高5倍以上,并且操作简便、快速,能在2 h内完成一次提取过程,极大地缩短了时间。以GS115中的内源AOX基因为目的基因,对提取的基因组进行PCR检测和Southern杂交检测,进一步验证了基因组的质量。因此,本文建立了一种简便、快速、经济而高效的酵母基因提取方法。     

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.     

Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)     

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 micro GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.