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Efficient expression of a foreign protein product by the yeast Saccharomyces cerevisiae requires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromere plasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, even in a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that a maximum of 4-6 centromere plasmids per cell can be stably partitioned at cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.

A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.

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