1. The rate of protein synthesis changes very little during the first 2-3 h (S phase) of the nuclear division cycle in plasmodia of Physarumpolycephalum and then increases continuously during G 2 phase, so that by the end of the cycle the rate has doubled relative to that in S phase. Protein synthesis appears to continue during mitosis.2. Fractionation of extracts of plasmodia, labelled with [3H]lysine for 1 h, by two-dimensional electrophoresis indicated that most if not all proteins are synthesised throughout the nuclear division cycle. However, two metabolically stable polypeptides, the synthesis of which occurs predominantly in G 2 phase, were detected.3. Using a double-labelling procedure, the differential rates of synthesis of 30 relatively abundant polypeptides were measured in relation to the nuclear division cycle. As a group, their differential rates of synthesis increase during the cycle so that their actual rates of synthesis increase 4-6-fold. This implies that their synthesis is regulated over and above any simple change due to a doubling in the number of genes during S phase.Growth of the plasmodial phase of the myxomycete, Pl~ysurum polycephalum, occurs without cell division, and the nuclei in the common cytoplasm divide with a high degree of synchrony [l]. There is no detectable delay (or GI phase) between the completion of nuclear division and S phase, which lasts 2 -3 h [2,3]. The remainder of the cycle (6 -7 h) is occupied by G2 phase; mitosis itself only lasts 20-30 min.The natural synchrony characteristic of the plasmodial phase of Physarum makes it a useful organism for the study of biochemical processcs in relation to nuclear division. The synthesis of protein between successive nuclear divisions has previously been determined by pulse-labelling with ["S]-methionine [4] and by an indirect reference to the synthesis of total nucleic acid [5]. However, unequivocal interpretation of pulse-labelling experiments is difficult, and the number of measurements required in the other study led to considerable experimental error in the values calculated for the increment in protein.Initially it was considered [5] that the isotope dilution method, developed for the study of metabolically stable nucleic acids [3], would not be directly applicable to the analysis of protein synthesis, because of the largely unknown extent of protein turnover. Further reflection suggested that amino acid labelling could be used provided that sufficient growth in tinlabelled medium was allowed before beginning specific activity measurements. If the chase period is sufficiently long, then radioactivity in proteins with a short half-life will be progrcssively lost and will accumulate in proteins that are either completely stable during growth or have a lifetime that is long by comparison with the intermitotic period. Once radioactivity is confined to metabolically stable proteins, the decrease with time of the specific activity of the total protein of the cell will be a simple index of net synthesis.In this paper we present...

A mutant of Escherichia coli with a decreased growth efficiency has been investigated. The results of growth studies with different substrates and of measurement of P/O ratios in membrane preparations suggest that the strain is defective in the ability to couple synthesis of ATP to electron transport. I N T R O D U C T I O NEscherichia coli strain 15-28 is a mutant containing an abnormally high concentration of the immediate precursor to the 50 s ribosomal subunit (MacDonald, Turnock & Forchhammer, 1967). The strain grows much less quickly than the wild-type and a genetic analysis (Turnock, 1969) showed that at least two mutations are responsible for its complex phenotype. One of these mutations (b-; Turnock, 1969) confers upon the cell an altered response to the antibiotic streptomycin and a decreased efficiency in the utilization of the growth substrate, compared to the parent strain. The latter property is examined in this paper; the results obtained suggest that the mutation reduces the efficiency of energy metabolism, probably that associated with oxidative phosphorylation. The significance of this finding and ways in which other mutants defective in oxidative phosphorylation might be selected are discussed. METHODSStrains of Escherichia coli. The two strains, b-and b f , used in tlus paper were obtained by mating Hfr 15-5 with strain 1 5 -2 8~ (Turnock, 1969). They both carry the same str and thy alleles, but do not contain the mutation responsible for the high concentration of ribosome precursor in strain 15-28.Media and growth conditions. Unless otherwise stated, liquid cultures were grown with aeration by shaking at 37'. Culture population densities were measured spectrophotometrically at 450 nm with a Gilford microsample spectrophotometer (light path, I cm). The minimal salts medium used was that of Turnock (1969), supplemented with thymine (10 mg/l) and glucose (2 g/l) or carbon source as indicated. For the preparation of cell-free extracts the bacteria were grown in minimal medium with glucose as carbon source supplemented with I % vitamin-free Casamino acids.For growth under anaerobic conditions, inocula were grown in screw-capped bottles filled to the neck with medium and diluted into growth flasks bubbled with95 % N2, 5% CO,. When NO3-(as 0-1 % KNO,) was used as the terminal electron acceptor, the flasks were bubbled with N,, and NaHCO, (25 mM) included in the medium (Cox et al. 1970).Growth yields. The extinction of the culture, measured at 450 nm, is proportional to the cell mass (Schaechter, Maalere & Kjeldgaard, 1958) and so this parameter was employed to

1. Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent K, of 29 pM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine.2. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides.3. As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C-2 of the pyrimidine ring to COZ. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.The plasmodia1 form of the true slime mould Physarum polycephalum has been used extensively for the study of nucleic acid metabolism during the synchronous nuclear division cycle. However, although many investigations have involved pulse-labelling with radioactive precursors (reviewed by Grant [l]), little is known about the mechanism of uptake of such precursors or of their subsequent metabolism, apart from the appearance of at least some of the radioactivity in nucleic acids.The uptake of nutrients by the plasmodial form of P. polycephalum is most conveniently studied in suspension cultures of microplasmodia. However, a major use of the organism is the analysis of biochemical processes in relation to the synchronous nuclear division cycle in surface plasmodia. Accordingly, we have examined the uptake and utilisation of uridine both by microplasmodia in liquid culture and by surface plasmodia growing on a filter paper support in contact with liquid medium [2]. Our results show that uridine is catabolised, as well as being used as a precursor for nucleic acid synthesis, so that continuous labelling of nucleic acids over an extended period of time is not possible without refeeding. Uptake of uridine by microplasmodia occurs via a specific, saturatable transport system that is inhibited competitively by other nucleosides. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the medium and the uptake is not inhibited by other nucleosides, suggesting a different mechanism of uptake (simple diffusion and/or pinocytosis) from that observed in microplasmodia. MATERIALS AND METHODS Growth of PlasmodiaStock cultures of microplasmodia of strain CL [3] of P. polycephalum were maintained in liquid culture at 26 "C both in complex medium containing mycological peptone and in medium containing casamino acids [4].Surface plasmodia were prepared from microplasmodia in the exponential phase of growth in mycological peptone and were grown on the same medium on Schleicher and Schuell chromatography paper no. 576 supporte...

1. The binding of putrescine and spermidine to free 70-S ribosomes has been studied by equilibrium dialysis a t 4" C in the presence of physiological concentrations of K+ and Mg2+. The number of binding sites for each polyamine is approximately 800, and the association constants for putrescine and spermidine are 80 and 500 mM-l, respectively. I n the case of spermidine there is evidence that a small fraction of the sites (3-50/, of the total) have an affinity for spermidine that is about 10 times higher.2. Competition experiments suggest that the two polyamines bind a t closely related sites on the ribosome.3. The binding sites for spermidine are distributed in proportion between the two ribosomal subunits.4. The 820, of free 70-S ribosomes (62 8 ) is unaffected in the presence of physiological concentrations of the two polyamines. 5. The effect of pressure in promoting dissociation of 70-S ribosomes to subunits is reduced by putrescine and spermidine.Polyamines are found in a wide variety of organisms and they have been implicated in a considerable number of biochemical reactions. At physiological pH values their primary and, when present, secondary amino groups are protonated and particular attention has been paid to the possibility of ionic interaction with the negatively charged phosphate groups of nuclcic acids (reviewed in [1,2]). However, understanding of their precise role is still meagre.Escherichia coli contains putrescine (1,4-diaminobutane) and spermidine (1-aminopropyl-1,4-diaminobutane). Small amounts of the monoacetylated derivatives of both polyamines are also present [3]. Their pathways of biosynthesis have been elucidated [I], and E. coli also possesses active uptake systems for both molecules [4]. I n 1960 Cohen and Lichtenstein [5] showed that a large fraction of the polyamines of E . coli could be isolated in association with the ribosomes, and that spermidine in particular could substitute for Mg2+ in preserving the structure of the 70-5 particle. However, it subsequently became clear that the concentrations of mono-and divalent cations in the buffer used for the fractionation of extracts determine the proportions of the polyamines that remain associated with the riboso-31.

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