J. Cell Set. 55, 51-67(1982) 51 Printed in Great Britain © Company of Biologists Limited 1982 POLY(A)+ RNA POPULATIONS, POLYPEPTIDE SYNTHESIS AND MACROMOLECULE ACCUMULATION IN THE CELL CYCLE OF THE EUKARYOTE CHLORELLA P. C. L. JOHN, C. A. LAMBE*, R. McGOOKINf, B. ORR AND M. J. ROLLINS Department of Botany, The Queen's University, Belfast BTy iNN, U.K. SUMMARY Synchronous cultures of Chlorella, that were obtained with minimum metabolic perturbation by centrifugal selection, reveal that progress through the cell cycle requires no change in the poly(A)+ mRNA population, although changes do occur during nutritional adaptation. Of the abundant soluble proteins, 93 % are synthesized continuously through the cell cycle and those that are discontinuous show similar patterns in control cells. The synthesis of proteins is compared with parallel studies of accumulation of enzyme activity and it is shown that there is no discrepancy in their pattern of accumulation when both are studied under the same culture conditions. The eukaryote cell cycle can allow stable relative rates of synthesis of most proteins and balanced rates of accumulation of most enzyme activities. Macromolecule classes differ in their rates of accumulation throughout the cell cycle: total RNA increases + linearly, poly(A) RNA accumulation is restricted to Gt phase, but total protein accumulation accelerates smoothly through Glt S and mitosis phases, pausing at cytokinesis. There is no evidence that the cell cycle requires an extensive programme of differential enzyme synthesis. The cycle can therefore proceed with minimum disturbance of metabolism required for growth. INTRODUCTION Most reports of enzyme activity in the cell cycle have described discontinuous patterns of increase. Such periodicity is particularly clear in synchronized populations of algae such as Chlorella (John et al. 1973; Schmidt, 1974; Lorenzen & Hess, 1974), and has been the subject of numerous reports in synchronous cultures of Saccharomyces cerevisiae (see reviews by Mitchison, 1969; Halvorson, Carter & Tauro, 1971). Paradoxically, individual proteins in yeast have shown continuous synthesis when non-synchronous cells were pulse-labelled and then segregated into phases of the cycle (Elliott & McLaughlin, 1978). It has therefore been uncertain whether there is a programme of biochemical development extending throughout the cell cycle. • Present address: Biochemistry Group, Engineering Science Division, Harwell, OXi 1 oRA, U.K. t Present address: Department of Botany, The King's Buildings, Mayfield Road, Edinburgh, EH9 3JH, U.K. 52 P. C. L. John, C. A. Lambe, R. McGookin, B. Orr and M. J. Rollins The present study rejected the use of synchronization by periodic illumination, which is commonly used to study the cell cycle of unicellular algae (John et al. 1973; Lorenzen & Hess, 1974), because, although periodic illumination does yield highly synchronous populations of cells, we have recently compared such cultures with others in which cells experienced continuous illumination and a constant degree of self-shading, and we have detected that synchronization that involves changes in illumination initiates metabolic adaptations that are not part of the cell cycle. Change in the amount of light available to individual cells causes fluctuations in photosynthetic capacity, in levels of starch, in rates of respiration and in the accumu- lation of enzyme activities that occupy a whole cell cycle (John, Lambe, McGookin & Orr, 1981). Similar distortions of enzyme accumulation caused by starvation and by osmotic stress were noted when sucrose density-gradient centrifugation was used to select small cells of Schizosaccharomyces pombe for synchronous culture (Mitchison, 1977). In the present study synchronous cultures were prepared by selecting small cells by continuous-flow centrifugation from an asynchronous population that was growing under constant environmental conditions. Care was taken that the selected cells were cultured at the same turbidity as they were in the parent culture, since a change in culture density would alter the amount of light reaching individual cells and so alter the growth rate. The potential hazard of such changes in growth rate for the study of enzyme synthesis is indicated by fluctuations in the rate of accumulation of four enzymes in Chlorella, following a 40% change in culture density (John, Cole, Keenan & Rollins, 1980). MATERIALS AND METHODS Cell culture Chlorella strain 211-Sp was cultured in mineral salts medium under conditions of illumi- nation and aeration that have been described previously (McCullough & John, 1972). For turbidostat culture a control system similar to that of Myers & Clark (1944) was used. An imbalance between a photocell receiving light through the culture and a reference photocell allowed an inflow of fresh medium, which stabilized culture turbidity. Medium inflow Was continually monitored by the automatic recording of liquid level in the medium reservoir. The extent of culture dilution up to each sample time was calculated, taking into account periods when the culture was recovering its volume after sampling and periods when the culture was overflowing (Herbert, Elsworth & Telling, 1956). Data presented in Figs. 1 and 2 have been corrected for culture dilution. They therefore provide a direct indication of growth and show patterns of accumulation that would have been observed in batch culture, if effects of increasing turbidity and increasing mutual shading could have been eliminated. Cell selection Small cells were selected from asynchronously dividing culture by continuous-flow centri- fugation (Lloyd, John, Edwards & Chagla, 1975). A nylon rotor was made in the form of a shallow cup of 90 mm diameter and 25 mm deep. The sides tapered inwards leaving an opening of 76 mm and the inner face of the rotor contained 11 semi-circular pockets 6 mm deep, into which the larger cells sedimented. Smaller cells passed out of the rotor, over the lip of the rotor cup with the overflowing medium, and were collected in an enclosing dish, from which they drained and were then aerated to await refractionation or inoculation into culture. mRNA and proteins in the cell cycle 53 Cell number Cell density was determined using a Coulter Counter BZ. Protein and DNA Protein was estimated in extracts of broken cells after precipitation with 5 % (w/v) tri- chloracetic acid and DNA was estimated by diphenylamine reaction, as described previously (McCullough & John, 1972). RNA isolation and estimation RNA was extracted in the presence of RNase inhibitors and was estimated by measuring absorbance at 260 nm. Poly(A)+ RNA was purified by binding to oligo(dT)-cellulose and determined by measuring absorbance at 260 nm. Both RNA fractions were prepared as described previously (Lambe & John, 1979). Estimation of relative poly(A)+ RNA levels by hybridization with [*H]poly(U) followed the procedure of Fraser & Carter (1976). Cell-free protein synthesis The in vitro protein synthesizing system isolated from wheat germ was employed, as described by Roberts & Paterson (1973), with endogenous protein synthesis reduced below levels detectable by autoradiography, by prior treatment with Ca1+-dependent nuclease (Pelham & Jackson, 1976). The rate of protein synthesis was linear with time for 1 h and with up to 1 fig of poly(A)+ RNA added. In the assays performed for Figs. 3 and 4, 0-5 fig poly(A)+ RNA was added and incubation was for 1 h. Electrophoresis One-dimensional electrophoresis in the presence of sodium lauryl sulphate was performed in a 15 % (w/v) acrylamide gel according to the method of Laemmli (1970). Two-dimensional separation, with isoelectric focusing followed by electrophoresis in the presence of sodium lauryl sulphate, according to the method of O'Farrell (1975), was used to fractionate 20 fig samples of extracted protein for the study of in vivo protein synthesis. RESULTS A major advantage of preparing synchronous cultures by cell selection is that control experiments can be performed to test for the effects of synchronizing. Fig. 1 shows a control experiment in which asynchronously dividing cells, held at constant turbidity and illumination, were at time o subject to continuous-flow centrifugation, but at a reduced rotor speed so that cells at all stages of the cell cycle remained in the supernatant fraction. After centrifugation the cells were transferred into a smaller vessel, to mimic the treatment of selected cells, and were grown at the same constant illumination as in the larger parent culture. The similarity of environment before and after selection is reflected in the unaltered rates of increase in each class of macromolecules and in cell number (Fig. 1). There is therefore no evidence that changes in the rate of macromolecule accumulation are caused by the cell selection procedure. The continuous-flow centrifugation procedure can be used to select a homogeneous population of small daughter cells, which form a synchronously dividing culture (Fig. 2) in which S phase is completed between 5 h and 10 h and subsequent release of daughter cells occurs between 12 h and 18 h. Cell-cycle events are shown 5I1 54 P. C. L. John, C. A. Lambe, R. McGookin, B. On and M. J. Rollins 20 20 10 _ 3 10 1 3 O 30 r- I 5 -| o X 20 & < £ z 3 i 3 a 10 5 20 r 1 40 a £ 10 - a < 20 ax s> if 106I I3 _L -12 -6 0 6 12 18 24 Time of growth relative to mock selection (h) Fig. i. The effect of cell selection procedures imposed at time o, on the increase in cell number (O); the accumulation of DNA (A); total protein (•); total RNA (•); and poly(A)+ RNA (A) in synchronously dividing Chlorella. A zo-1 culture was grown at 25 °C in a narrow trough-like vessel with sides 1200 mm x 236 mm and a breadth of 70 mm. Illumination was provided by two banks of warm-white fluorescent strip-lights giving i4O/<Einsteins M~2 S"1 at 400-700 nm onto both sides of the vessel.