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...
1. The degree of homology between ribosomal RNA isolated from microplasmodia and amoebae of the slime mould Physarum polycephalum has been determined by competitive hybridisation of the RNA from the two sources to homologous DNA in solution. The extent of competition was measured both by hybridisation to saturation and by following the kinetics of hybrid formation.In each case competition was found to be 100 %, indicating that the ribosomal RNAs from the two, quite different vegetative forms of the organism exhibit a high degree of homology and are probably transcribed from the same genes.2. The relationship between the amount of nuclear DNA that codes for ribosomal RNA (rDNA) and ploidy has been investigated in three strains of P. polycephalum which exhibit a 1 : 2 : 5 variation in the amount of DNA per nucleus. Ribosomal RNA saturation values were determined by hybridisation to DNA isolated from prophase nuclei of plasmodia. The proportion of rDNA was found to be constant at 0.16-0.18% of the total genome in the three strains.The genes coding for ribosomal RNA in the slime mould Physarum polycephalum have been shown to be located in the nucleolus by cell fractionation [l], light microscopic autoradiography [2] and electron microscopic autoradiography [3]. Unlike bulk DNA, rDNA is replicated throughout the mitotic cycle [l, 4,5] with the exception of the first hour after mitosis [l]. Measurements of the amount of rDNA have varied from 0.1 % to 1.3 % [5,6,41] of the nuclear DNA.In this paper we have applied RNA-DNA hybridisation techniques to investigate the following two basic questions concerning rDNA in P. polycephalum. a) Are the same genes involved in rRNA transcription in the plasmodia1 and the amoeba1 stages of the life cycle? b) Does ploidy have any effect on rRNA gene dosage in plasmodia?The latter question has been investigated in several other organisms. Species of the genus Nicotiana have Abbreviations. rDNA, that fraction of DNA which codes for rRNA; standard saline citrate, 0.15 M NaCl plus 0.015 M sodium citrate, 5 mM EDTA, pH 7.0; Napi, equimolar mixture of NaH,P04 and Na2HP04, e.g. 0.24 M Napi is equivalent to 0.12 M NaH2P04, 0.12 M Na,HPO,; T,, the mean temperature of dissociation measured optically; Tm,i, the mean temperature of strand separation.Enzymes. Ribonuclease TI (EC 3.1.4.8); ribonuclease A (EC 3.1.4.22); a-amylase(EC 3.2.1.1); fi-amylase (EC 3.2.1.2); pronase (EC 3.4.21.4 + 3.4.24.4). similar absolute numbers of rRNA genes despite a two-fold variation in chromosome number [7]. On the other hand, in a polyploid series of plants (haploid to hexaploid) of Datura innoxia produced by culture of pollen grains in vitro, the proportion of rDNA has been shown to be constant [8]. Investigations of this kind, however, are only strictly meaningful if either (a) the proportion of rDNA is constant throughout the cell cycle, i.e. bulk DNA and rDNA are replicated at the same time, or (b) determinations of gene dosage are carried out at a fixed point in the cell cycle necessitating synchronous cultu...
of sedimentation coefficient 97 S, were isolated from vegetative cells and cysts. Both polyribosoines and ribosomes dissociated in 0.3 M-KCl to particles of sedimentation coefficients 66, 53 and 40 S, and a slowly sedimenting particle whose sedimentation coefficient was not measured. RNA was extracted from the particles and fractionated. The 53 S and 40 S particles are the ribosomal subunits; the 97 and 66 S particles contained RNA characteristic of both subunits. I N T R O D U C T I O NAcanthamoeba castehzii* is a Hartmanellid amoeba particularly suited to metabolic and structural studies as it can be readily grown in axenic culture. The properties of the ribosomes of this organism are of particular interest in the light of its unusual rRNA. The RNA (18 S RNA) from the smaller ribosomal subunit is anomalously large (mol.wt 0.9 x lo6; Loening, 1968a) and the large RNA (26 S RNA) molecule from the larger ribosomal subunit contains in vivo a single-strand break, with the result that, upon denaturation, two RNA species are derived from it (Stevens & Pachler, 1972).These properties, unusual among eukaryotic organisms, may be reflected in anomalous properties of the ribosome. Stevens & Pachler (1972) described a method for obtaining a ribosomal pellet from the vegetative cells but presented no data on the properties of the ribosomes. Changes in ribosomal structure might also be associated with the morphogenetic cycle of the amoeba. M E T H O D SChemicals. Mycological Peptone was obtained from Oxoid Ltd, London, Tris base from Sigma Chemical Co, St Louis, Missouri, U.S.A., puromycin hydrochloride from the Nutritional Biochemicals Corporation, Cleveland, Ohio, U.S.A., and sodium tri isopropylnaphthalene sulphonate from Kodak Ltd, London. Nonidet P40 was a gift from Shell Chemicals Ltd, London. All other chemicals were obtained from British Drug Houses Ltd, Poole, Dorset, and were of AnalaR grade wherever possible. Acrylamide and methylene bis acrylamide (used for making polyacrylamide gels for electrophoresis) were recrystallized by the methods of Loening (1968 b).Growth of amoebae. Acanthamoeba castellanii NEFF was kindly supplied by Dr U. E. Loening of the Department of Zoology, University of Edinburgh. The organism was maintained by serial transfer in small flasks of 4 % (w/v) Mycological Peptone at room temperature. For growth, a sample (10 ml) of a stock culture was added aseptically to 250 ml of 4 % Mycological Peptone, supplemented with 0.9 % (w/v) maltose, in a I 1 flask * Referred to the genus Hartnzcmella in some earlier literature.
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