Synthesis of Ribosomal RNA during the Mitotic Cycle in the Slime Mould Physarum polycephalum

Abstract

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...

Synthesis of protein during the nuclear division cycle in Physarum polycephalum

“…Large surface plasmodia were grown as described previously [1,2]. Microplasmodia were labelled with [2:4C]uridine (10/AVI; 0.6 btCi ml -a) rather than [5,6-aH]uridine that was used in earlier work [2] ; the procedure for labelling was otherwise unchanged.…”

“…However, the isotope-dilution method can be applied indirectly by prelabelling the metabolically stable nucleic acids and then, in samples from a surface plasmodium, determining both the specific activity of the nucleic acid (S) and the amounts of nucleic acid (iV) and protein (P). Since S is proportional to N -1 [1], the relative accumulation curve for protein is given by S • N -1 • P. This paper describes the results obtained for the synthesis of protein during the mitotic cycle using this method.…”

“…We have previously used the isotope-dilution method to follow the synthesis of individual classes of nucleic acid (DNA and rRNA [1 ] ; tRNA and rRNA [2] ). Figure 1, in contrast, shows the pattern of accumulation of total nucleic acid between postfusion mitoses II and III in a surface plasmodium.…”

“…Another important advantage of the method is that it does not require all the samples taken in one experiment to be identical in size. The procedure has been used to derive the patterns of synthesis of DNA and rRNA [1] and tRNA [2] during the nuclear division cycle.…”

“…We have developed an isotope-dilution method [ 1 ] which can be used to study the synthesis of any macromolecule that is metabolically stable and which does not involve the difficulties of interpretation of pulse-labelling experiments. Another important advantage of the method is that it does not require all the samples taken in one experiment to be identical in size.…”