Co-ordination of macromolecular synthesis in Physarum polycephalum.

1. The object of this work was to investigate some aspects of the regulation of protein synthesis in a simple eukaryote, the slime mould, Physarum polycephalum. 2. Background - In bacteria, the RNA content per cell decreases as a linear function of decreasing growth rate. The amount of protein per cell also declines but in such a way that the rate of protein synthesis per unit rRNA is constant over a wide range of growth rates. Thus, in bacteria, ribosome efficiency is largely independent of growth rate. (1, 2 and 3) 3. Culture conditions - The steady state growth rate of P. polycephalum at 26°C was varied as follows: i Microplasmodia were grown in different axenic media i.e. as batch cutures ii The supply of one nutrient (glucose) was limited i.e. chemostat cultures. An approximately four-fold range of growth rates (? = 0,017 to 0,07 h-1 at 26°C ) was obtained by both methods. 4. Approach - Chemical assays were used to estimate the relative proportions of DNA (diphenylamine assay), RNA (absorption of nucleic acids at 260 nm) and protein (Folin assay). Extracts of total RNA were prepared and analysed for the proportion of rRNA. The rate of protein synthesis per ribosome, k, was calculated from the relative proportions of protein and rRNA at particular growth rates, ?, as described by Koch (4). Preparation and analysis of polyribosomes enabled the proportion of ribosomes actively engaged in protein synthesis to be estimated. Thus, the rate of protein synthesis per active ribosome, kr, could be calculated, 5. Results For microplasmodia maintained in batch culture, the RNA /DNA and RNA /protein ratios have a curvilinear relationship with growth rate, ?. Protein /DNA ratio hardly varies with ?. ii The two culture methods generated closely similar data except at low growth rates (?< 0.025 h-1) where microplasmodia in chemostat culture had a consistently higher proportion of RNA. iii About 85% of the RNA was found to be ribosomal (? > O.O5 h-1). The variation with growth rate and culture method was small. iv For microplasmodia in batch culture, the ribosome efficiency, k, fell steadily from 108 (? = 0.07 h-1) to 64 (?< 0.017 h-1 ) amino acids min-1 On comparing these values with those obtained from chemostat cultures, the only significant differences were observed at slow growth rates (? < 0.025 h-1) where chemostat cultures generated lower ribosome efficiencies.( k =44 amino acids min-1 ; ?=0.017 h-1). V Analysis of polyribosome preparations showed considerable variation between the two culture methods. For batch cultures, the polysome content changed very little over the range of growth rates studied. For microplasmodia maintained in chemostat culture, however, the polysome content showed a linear dependence on ? ranging from 45% (? = 0.017 h-1) to 76% (? = 0.07 h-1). vi In the case of batch culture data, the variation of kr with ? was qualitatively similar to that observed for k. It is not possible to say categorically whether the peptide chain growth rate at a particular ? is the same for the two culture methods. This uncertainty arises from the large monosome population in many chemostat culture experiments. Vii Possible interpretations for the observed variation of kr with ? are discussed. It is conceivable that the peptide chain elongation rate decreases with decreasing growth rate, although protein turnover could, equally well, account for the results. Viii The difference between the two culture types is further emphasised by 'shift- up' experiments. In batch cultures, the rate of RNA synthesis started to increase almost immediately after the shift whereas a lag of 5 to 6 hours was observed in chemostat cultures of the same pre-shift growth rate. 6. Data from chemostat cultures differ in several respects from those obtained from batch cultures especially at low growth rates. These differences are discussed in relation to early stages of differentiation of microplasmodia to spherules. References 1. Dennis, P.P. and Bremer, H. (1974) J. molec. Biol., 84, 407-422. 2. Forchhammer, J. and Lindahl, L. (1971) J. molec. Biol., 55, 563-568. 3. Maaloe, o. and Kjeldgaard, N.O. (1966) in Control of Macroraolecular Synthesis. New York: W.A. Benjamin. 4. Koch, A.L. (1970) J. theor. Biol., 28, 203-231.