The Cell Cycle of the Budding Yeast Sterigmatomyces halophilus: Culture Fractionation by Zonal Centrifugation and the Accumulation of DNA, RNA and Protein

Abstract

Sterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed, remote from the mother cell, on fine projections called sterigmata. Some fundamental properties of the cell cycle have been explored by separating cells from an exponentially growing culture into size, and thus age, classes by density-gradient centrifugation. Rate separations on high capacity, high resolution, equivolumetric gradients of sucrose, or, alternatively, isopycnic separations on gradients of Urografin revealed consistent and reproducible patterns of accumulation of DNA, RNA and protein through the cell cycle. Total DNA accumulation was stepwise, synthesis occurring late in the cycle, whilst protein accumulated continuously with no evidence for the discontinuities reported in some other lower eukaryotes. Total RNA accumulation, measured either colorimetrically or by long-term incorporation of radioactively-labelled uracil was transiently elevated early in the cycle and then accumulated continuously. A mathematical analysis of the volume distributions of the cells in fractions from the gradients showed that there is a hyperbolic relationship between cell age and size but that, to a first approximation, measurements of cell size (and density) are direct measures of age. The results are discussed with reference to (1) the unusually high buoyant density of this yeast, (2) the resolution of zonal cell separation methods and (3) macromolecular accumulation in the cell cycles of other eukaryotic micro-organisms.

The Cell Cycle of the Budding Yeast Sterigmatomyces halophilus: Oscillations in the Amounts and Activities of the Terminal Components of the Respiratory Chain

“…Sterigmatomyces halophilus CBS 4609 was maintained on a solid, complex medium and grown in a defined liquid medium with glucose as the growth-limiting substrate as described previously (Salmon & Poole, 1980a). Exponentially growing cultures were harvested by passing the culture through an MSE continuous action rotor and resuspending the cells in a small volume of 3% (w/v) NaCl (Salmon & Poole, 1983).…”

“…Fractionation of the cell population into various size classes, representing successive stages in the cell cycle, was by isopycnic-zonal centrifugation through Urografin gradients (Salmon & Poole, 1983). Fractions were collected, and the cells harvested and washed as described previously (Salmon & Poole, 1983) and resuspended in 50 m~-Tris/H$O, buffer (pH 7.2).…”

“…These were performed using a Coulter Counter model ZBI fitted with a 50 pm aperture probe and Channelyzer ClOOO as described by Salmon & Poole (1983).…”

“…The total protein content of whole cells was determined as described by Salmon & Poole (1983). Tris interference was not as marked in this assay, but corrections were made if the final concentration of Tris exceeded 0.1 5 mM.…”

“…Additionally, the activities of two marker enzymes (Lloyd & Cartledge, 1974) for other cellular membraneous systems (catalase for peroxisomes and acid phosphatase for lysosomes) were measured. Sufficient material for such studies was provided by large-scale zonal fractionation of exponentially growing cultures into different density and size classes (Salmon & Poole, 1983). Fourth-order finite difference analysis of the complex cytochrome spectra was used to resolve and quantify the individual components, some of which accumulate continuously, whilst cytochrome c and its immediate electron acceptor, cytochrome aa3, oscillate in phase with cytochrome c oxidase activity.…”