Kinetics of the cell cycle of Saccharomyces cerevisiae.
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Bromodeoxyuridine↗
Biomedical subjects
F Srienc
Publications and source records attributed to F Srienc.
A statistical analysis of flow cytometric determinations of phagocytosis rates.
Uptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, ... particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.
Analysis of Variance↗
Dynamics of activation of a galactose-inducible promoter in Saccharomyces cerevisiae.
We have investigated the dynamics of accumulation of the Escherichia coli beta-galactosidase (beta-gal) under the control of a promoter containing the galactose-inducible upstream activating sequence (UASG) in single Saccharomyces cerevisiae cells. The accumulation of beta-gal in single cells following the addition of the inducer, galactose, was determined using an in situ combined DNA and immunofluorescent stain in conjunction with flow cytometry. Two strains were studied, D603/2i, which has two copies of the galactose-inducible fusion gene integrated into its genome, and D603/pLGSD5, which carries a 2 microns-based plasmid containing the fusion gene. Flow cytometry results indicate that accumulation of beta-gal within the first three hours following the addition of galactose is dependent on cell cycle position. Two proposed mechanisms explaining this observed behavior are (1) the cell-cycle-dependent synthesis of the fusion protein or (2) the unequal partitioning of the protein at cell division between mother and daughter cells.
Cell Cycle↗
Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan.
Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used.
Animals↗
A discrete, stochastic model for microbial filter feeding: a model for feeding of ciliated protists on spatially uniform, nondepletable suspensions.
Suspension-feeding ciliates, either bacteriovorous or planktonic, are adapted to feed on particulate food matter of size much smaller than their own size. These microorganisms collect their prey by generating water currents that draw prey toward their capture surfaces. Under such conditions food particles are treated in bulk, and captures of individual food particles from a suspension by individual single-celled organisms are discrete events that occur at random intervals of time. Each such event is followed by a sequence of additional events that also occur at random intervals of time. This sequence culminates in the incorporation of the digestible portion of the food particle into the cell's cytoplasm and the expulsion of the indigestible portion from the cell. In theory, the rate of the overall ingestion-digestion process can be limited by the passage of particles through any stage of this sequence of events. In this paper, we assume that only the initial events in the sequence, those that occur in the oral region of the cell, limit the rate of the ingestion-digestion process, and we develop a discrete, stochastic model of filter feeding based on that assumption. We use the model to show how advanced instrumentation, such as flow cytometry, can be used to measure parameters of the model and also to answer a number of important questions about the mechanism of filter feeding. We show also how the model can be applied to nonhomogeneous cell populations for which parameters of the model are distributed.
Animals↗
Flow cytometric study of hybridoma cell culture: correlation between cell surface fluorescence and IgG production rate.
Flow cytometry was applied to measure the fluorescence of IgG molecules on hybridoma cell surface stained with FITC-conjugated anti-mouse IgG F(ab')2. Using light scattering to exclude the dead cell population from the analysis, the surface fluorescence intensity allows for the differentiation between producing and nonproducing cells. A linear correlation has been found between the intensity of mean surface fluorescence of the cell population and the specific antibody production rate.
Animals↗
Quantitative immunofluorescence in single Saccharomyces cerevisiae cells.
We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level. Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.
DNA, Bacterial↗
Feeding, growth, and reproduction of ciliate microorganisms. An engineering view.
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Animals↗
Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations.
A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.
Flow Cytometry↗
Studies of host-plasmid interactions in recombinant microorganisms.
Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.
DNA, Recombinant↗
Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae.
We have used a set of deletion mutations in the ARS1 element of Saccharomyces cerevisiae to measure their effect on chromosome stability. This work establishes the previously proposed existence of three domains in ARS1. Domain C, which we have previously inferred, but not proved, to be a part of ARS1, is now established. In addition, we show that increasingly large deletions of the domain have increasingly large effects, which was not realized before. Furthermore, we have provided the first positive evidence for the central importance of a 14-base-pair core sequence containing the ARS consensus element by showing that it has the ability to act as a replicator on a plasmid containing no other ARS1 flanking sequence. The method of analyzing plasmid stability used in our study employs a novel and sensitive flow cytometry assay for beta-galactosidase. We discuss ways in which flow cytometry, based on this assay, could be generalized beyond its particular application in this work to studying other aspects of the cell biology of yeast and higher cells. The actual flow cytometry method will be described in detail elsewhere.
Chromosomes↗
Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae.
DNAs that contain specific yeast chromosomal sequences called ARSs transform Saccharomyces cerevisiae at high frequency and can replicate extrachromosomally as plasmids when introduced into S. cerevisiae by transformation. To determine the boundaries of the minimal sequences required for autonomous replication in S. cerevisiae, we have carried out in vitro mutagenesis of the first chromosomal ARS described, ARS1. Rather than identifying a distinct and continuous segment that mediates the ARS+ phenotype, we find three different functional domains within ARS1. We define domain A as the 11-base-pair (bp) sequence that is also found at most other ARS regions. It is necessary but not sufficient for high-frequency transformation. Domain B, which cannot mediate high-frequency transformation, or replicate by itself, is required for efficient, stable replication of plasmids containing domain A. Domain B, as we define it, is continuous with domain A in ARS1, but insertions of 4 bp between the two do not affect replication. The extent of domain B has an upper limit of 109 bp and a lower limit of 46 bp in size. There is no obvious sequence homology between domain B of ARS1 and any other ARS sequence. Finally, domain C is defined on the basis of our deletions as at least 200 bp flanking domain A on the opposite side from domain B and is also required for the stability of domain A in S. cerevisiae. The effect of deletions of domain C can be observed only in the absence of domain B, at least by the assays used in the current study, and the significance of this finding is discussed.
Base Sequence↗
Kinetics of product formation and plasmid segregation in recombinant microbial populations.
Experimental and mathematical analysis of productivity of cultures containing recombinant plasmids is based in this work on the following paradigm: molecular controls leads to single cell kinetics leads to cell population dynamics leads to reactor productivity. Mathematical models have been developed for replication control of the lambda dv plasmid and for efficiency of the lac promoter-operator based on the molecular control mechanisms of these systems in Escherichia coli. A special and important attribute of these models is their ability to describe quantitatively a wide range of genetic effects as well as environmental influences on molecular control function. Equations describing plasmid maintenance and distribution in growing cell cultures have been determined based on the population-balance equations applicable to a segregated culture model. The washout dilution rate for continuous cultivation of plasmid-containing Saccharomyces cerevisiae in selective medium is given in terms of single-cell division-cycle parameters and plasmid copy number for one single-cell model of plasmid replication and segregation. A new experimental method based on flow cytometry for rapid characterization of heterogeneity of single-cell accumulation of a plasmid gene product is also described. Generalization of these methods and of the overall strategy should provide a useful framework for synthesis of biological and engineering principles and methods to optimize organisms and processes based on recombinant DNA technology.
Bacteria↗
Production of two phase polyhydroxyalkanoic acid granules in Ralstonia eutropha.
To synthesize layered granules consisting of selected phases of polyhydroxybutyrate (PHB) homopolymer and PH(B-co-V) copolymer, Ralstonia eutropha was grown on fructose and limited quantities (1 g/l) of valeric acid. Exhaustion of the valerate resulted in a carbon source shift and a shift in the composition of polyhydroxyalkanoate (PHA) being synthesized within the cell. The synthesis rates were 0.030 g PH(B-co-V)/l per h and 0.033 g PHB/l per h, giving a copolymer composition of 48% HV. The valerate was exhausted at approximately 12 h at a rate of 0.0894 g/l per h after which only PHB was produced through the remaining 12 h at 0.033 g PHB/l per h from the remaining fructose, which was utilized at a constant rate of 0.0861 g/l per h throughout all 24 h of the experiment. Differential scanning calorimetry (DSC) of isolated granules showed two glass transitions, confirming the presence of two distinct polymer phases within the layered granules. Transmission electron microscopic images stained with RuO4 revealed a heavily stained copolymer core within a lighter stained PHB shell, confirming the expected morphology of granule composition. Thus, biosynthesis can be exploited for the control of domain sizes in layered granules, potentially providing metabolic control over the physical properties of the resultant polymer.
Alcaligenes↗
Slit scanning of Saccharomyces cerevisiae cells: quantification of asymmetric cell division and cell cycle progression in asynchronous culture.
Slit scanning flow cytometry has been applied to the analysis of the cell cycle and cell-cycle-dependent events in Saccharomyces cerevisiae, yielding information on the low-resolution spatial distribution of cellular components in single cells of unperturbed cell populations. Because this process is rapid, large numbers of cells can be analyzed to give distributions of parameters in a given population. To study asymmetric cell division and cell cycle progression, forward-angle light scattering (FALS) signals together with fluorescence signals from acriflavine-stained nuclei have been measured in cells from exponentially growing yeast populations. An algorithm has been developed that assigns the position of the bud neck in the FALS signals so that both FALS and DNA signals can be analyzed in terms of the contributions from the mother cell and the cell bud. The data indicate that mother cell FALS, on average, remains constant while FALS due to the cell bud increases as a cell progresses through the cell cycle. By identifying mitotic cells and measuring their properties, we have found that the coefficient of variation for the distribution of FALS is smallest within the dividing cell population and largest within the newborn cell population, in accordance with the critical size control mechanism of yeast cell growth. The use of this experimental approach to provide data for statistical population models is discussed.
Acriflavine↗
Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.
An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.
Bromodeoxyuridine↗
Stability of producer hybridoma cell lines after cell sorting: a case study.
Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface-associated antibody contents to identify a significant non-producer cell fraction in a murine hybridoma cell line that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface-associated antibody content were isolated by cell sorting on the basis of surface fluorescence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultivated for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line during passaging and after a freeze-thaw cycle was investigated. This cell line was found to be highly unstable. A simple batch-growth model simulation was used as follows: (i) to demonstrate that nonproducers appear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy-chain gene and/or light-chain gene expression; (ii) to show that a high rate of conversion of producer hybridomas to nonproducer hybridomas cannot entirely explain the population dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 x 10(-5) h-1; (iv) to show that, for this conversion rate, the nonproducer cells' specific growth rates need only be 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cells are preferentially lost during cell freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analyzed and purified frequently to prevent overgrowth by nonproducers.
Animals↗
Tracking of individual cell cohorts in asynchronous Saccharomyces cerevisiae populations.
A novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture. The method is based on labeling of the cell surface with FITC-conjugated concanavalin A and detection of the single-cell fluorescence with flow cytometry after cell exposure to growth conditions. Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells. Analysis of the staining pattern over time permits the determination of the specific growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells. The procedure has been tested with yeast cell populations growing at different rates. The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals. It has been found that the apparent single-cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population. This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size.
Cell Cycle↗