Detection of promoter activity by flow cytometric analysis of GFP reporter expression

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.     

Flow cytometric studies during culture of Saccharomyces cerevisiae

Double-beam laser flow cytometry has been used to analyse samples from different Saccharomyces cerevisiae cultivation processes (batch and continuous culture). Using this technique, the biological parameters of cell size, and protein, RNA and DNA content can be determined. It is possible to study the budding behaviour from the cell size distribution as well as from DNA analysis. In oscillating continuous yeast cultures, flow cytometry studies were performed in addition to on-line NADH measurements. The effect of cell recycling on yeast metabolism was also the object of cytometric studies. Structured biological data can be obtained by this analytical technique, and the effect of different cultivation conditions on cell biology can be determined.     

Alpha-agglutinin expression in Saccharomyces cerevisiae

A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells. The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells. Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.     

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.     

"Active" one-carbon generation in Saccharomyces cerevisiae.

A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred.     

Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae

The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products.     

Immunofluorescent quantification of tyrosine phosphorylated proteins by flow cytometric analysis

To measure the quantity of tyrosine phosphorylated proteins in cells, we have used a flow cytometry technique with fluorescein isothiocyanate-labeled anti-phosphotyrosine antibody (FITC-PY mAb). The analysis was applied to the phosphotyrosine titration and showed an optimum amount of FITC-PY mAb (30g/1×10cells). The staining specificity of our assay was tested by the addition of exogenous competitors, showing a specific inhibition by phosphotyrosine but not by phosphoserine, phosphothreonine, or tyrosine. The assay was also able to elucidate the inhibitory effect on tyrosine phosphorylation of genistein, a protein tyrosine kinase inhibitor. These results imply that immunofluorescent quantification assay using a flow cytometer could be a useful technique to determine the intracellular level of tyrosine phosphorylated protein.     

Staining and quantification of poly-3-hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry.

BACKGROUND: Poly [(R)-3-hydroxybutyric acid] (PHB) is a prokaryote storage material for carbon and energy that accumulates in cells under unbalanced growth conditions. Because this class of biopolymers has plastic-like properties, it has attracted considerable interest for biomedical applications and as a biodegradable commodity plastic. Current flow cytometric techniques to quantify intracellular PHB are based on Nile red. Here, an improved cytometric technique for cellular PHB quantification utilizing BODIPY 493/503 staining was developed. This technique was then automated using an automated flow cytometry system. MATERIALS: Using flow cytometry, the fluorescence of Saccharomyces cerevisiae and Cupriavidus necator with varying PHB content after staining with BODIPY 493/503 and Nile red was compared, and automated staining techniques were developed for both cultures. RESULTS: BODIPY 493/503 staining had less background staining, higher sensitivity and specificity to PHB, and higher saturation values than did Nile red staining. The developed automated staining procedure was capable of analyzing the PHB content of a bioreactor sample every 25 min and measured the average PHB content with accuracy comparable to offline GC analysis. CONCLUSION: BODIPY 493/503 produced an overall better staining for PHB than did Nile red. When combined with the automated system, this technique provides a new method for the online monitoring and control of bioreactors.     

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes.     

Modeling and optimization of cloned invertase expression in Saccharomyces cerevisiae

The aim of this study is to determine the medium feeding strategy to maximize the invertase productivity of recombinant Saccharomyces Cerevisiae using a fed-batch mode of operation. The yeast contains the plasmid, pRB58, which contains the yeast SUC2 gene, coding for the enzyme invertase. The expression of this gene is repressed at high glucose levels. A Goal-oriented model is development to describe the kinetics of fed-batch fermentations. This simple model could quantitatively describe previous experimental results. A conjugate gradient algorithm is then used, in conjunction gradient algorithm is then used, in conjunction with this mathematical model, to compute the optimum feed rate for maximization of invertase productivity. The optimal feeding procedure results in an initial high cell growth phase followed by a high invertase production phase. (c) 1993 Wiley & Sons, Inc.     

An androgen-inducible expression system for Saccharomyces cerevisiae

A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.