The maintenance of self‐replicating plasmids in Saccharomyces cerevisiae: Mathematical modelling, computer simulations and experimental tests

Abstract

A distributive model has been constructed to describe the maintenance of the native 2 microns and 2 micron-based plasmids in the yeast Saccharomyces cerevisiae. This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability. A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out. These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence. The form chosen involves an amplification system in which the RAF gene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of the FLP gene. At the same time an upper limit (or 'ceiling') was imposed on the number of plasmid molecules able to replicate. Maternal bias was accommodated by 'tagging' a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or 'cleared') by the Rep1/Rep2 dimers. This final form of the model makes specific predictions about the stability of 2 microns and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations. The predictions on stability have been subjected to experimental test and results provide good support for the model.

Long-Distance Base Pairing in Flock House Virus RNA1 Regulates Subgenomic RNA3 Synthesis and RNA2 Replication

“…In this case, two fluorescent cell populations were apparent: ϳ75% of cells were brightly fluorescent, while the remainder were dimly fluorescent yet shifted relative to control cells lacking protein A. The dim population probably represents cells that recently lost one or both FHV expression plasmids, as plasmids segregate unequally in yeast (12,14,36,51). Consistent with this explanation, differential plating experiments indicated that ϳ20% of the total cell populations had lost the Leu ϩ or Trp ϩ phenotype.…”

Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

“…A fourth gene (D) on the 2m circular plasmid counteracts the regulatory function of the Rep1 and Rep2 proteins (24). A mathematical model for plasmid maintenance has been successfully applied in computer simulations and is supported by experimental data (33).…”

Biotransformation, Using Recombinant CYP450-Expressing Baker’s Yeast Cells, Identifies a Novel CYP2D6.10A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme

“…Extrachromosomal 2-micron (2 μ) plasmids are normally used for expression of heterologous (i.e., human) proteins in yeast . Unfortunately, a 2 μ plasmid can be maintained within yeast cells only when grown in selective synthetic defined (SD) minimal medium . Because such a medium lacks essential nutrients, a CYP gene expression cassette borne on a 2 μ plasmid would produce very little CYP protein/enzyme simply because of poor yeast cell growth.…”

“…21 Unfortunately, a 2 μ plasmid can be maintained within yeast cells only when grown in selective synthetic defined (SD) minimal medium. 22 Because such a medium lacks essential nutrients, a CYP gene expression cassette borne on a 2 μ plasmid would produce very little CYP protein/enzyme simply because of poor yeast cell growth. As an alternative, complete full YPD medium (containing yeast extract, peptone, dextrose/glucose), which cannot select for the presence of a 2 μ plasmid, could be used for short-term growth of cells which bear a CYP gene expression cassette on a 2-micron plasmid.…”