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1. An isotope dilution technique has been used to analyze the synthesis of metabolically stable nucleic acids during the mitotic cycle in surface plasmodia of the slime mould Physarumpolycephalum. Microplasmodia that had been labelled with [3H]uridine were used to prepare a surface culture, after a period of growth long enough to ensure that radioactivity was present only in tRNA, rRNA and DNA. The synthesis of rRNA or nuclear DNA during the growth of the surface plasmodium was then followed by measuring the specific activity of the nucleic acid, 2. Synthesis of rRNA during the mitotic cycle shows the following characteristics: (a) it is low during the immediate period of nuclear division, (b) synthesis is then continuous throughout interphasc and (c) the rate of synthesis increases 5 -6-fold between the beginning and end of interphase. These results are discussed in relation to the pattern of replication of the genes for rRNA. 3.Approximately 80 % of the nuclear DNA replicates during the first 90 min of the mitotic cycle; completion of replication, however, occupies the remainder of interphase.The plasmodia1 form of the Myxomycete, Physarum polycephalum, exhibits natural mitotic synchrony within a common cytoplasm [I], and it is an ideal organism in which to study the synthesis of specific macromolecules in relation to the nuclear division cycle. In surface plasmodia the inter-mitotic time is 8 -9 h; S phase begins immediately after nuclear division and occupies the first 2-3 h of the cycle Previous investigations of the synthesis of RNA during the mitotic cycle of P . polycephalum have been reviewed by Grant [5]. In general, pulse-labelling with [3H]uridine has been used to estimate the amount of RNA synthesis taking place, and the experiments are open to serious doubt in the absence of detailed, quantitative information about the mechanism of uptake of uridine, its subsequent metabolism and its relationship to endogenous synthesis of pyrimidine nucleotides. The amounts of the nucleoside triphosphates do vary during the mitotic cycle [6], and there is also the possibility, as has been demonstrated for animal cells [7,8], that the nuclear and cytoplasmic nucleotide pools may not be in rapid equilibrium with each other and that they may differ in their rate of labelling from exogenous nucleosides. In this paper, we describe an investigation of the synthesis of rRNA during the mitotic cycle of P . polycephalum, using an isotope dilution procedure that is not dependent upon comparison of the amounts of labelled nucleic acid precursor taken up at different times during the cycle. Our results show that the synthesis of rRNA is very low during the period of mitotic division and that the net rate of synthesis increases continuously throughout the remainder of the cycle, the change in rate between the beginning and end of interphase being 5 -6-fold. The significance of these results is discussed in relation to the pattern of replication of the genes for rRNA.Determinations of the duration of S phase in P . polycephalum...

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Four species of rapidly labelled, high-molecular-weight RNA have been extracted from partially purified nuclei of cultured sycamore cells. The molecular weights of these RNA species RNA is consistent with the sequence of reacttiom shown above, and has also demonstrated that methylation is confined to the rRNA sequences of the 45-8 RNA [6].I n other eukaryotes details of the processing of the precursors of rRNA are less well understood. The large 45-S precursor appears to be peculiar to mammals, and it has been suggested that the moleoular weight of the pre-rRNA is correlated with the evolutionary position of the organism [7]. A major difference between mammals and other groups of organisms lies in the large size of the excess RNA that is removed in the first maturation reaction (45-S + 41-8 in mammals). As far as higher plants are concerned a scheme for the synthesis and processing of mung bean cytoplasmic rRNA has been proposed [S], based on the molecular weights and rates of labelling of the various components separated by gel electrophoresis, on their base composition and on competitive hybridisation experiments. The stages of processing of the pre-rRNA are analogous (although with different molecular weights) to those suggested [3] for HeLa cell pre-rRNA.The amount of excess (non-ribosomal) RNA in the ikst precursor appears to vary considerably in plants, even between closely related species [9] and differences have also been reported in pre-rRNA isolated from M e r e n t tissues of the mme plant [S]. I n addi-

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...