Ribosomal RNA Genes in the Amoebal and Plasmodial Forms of the Slime Mould Physarum polycephalum

Abstract

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...

The Replication of Ribosomal DNA in Physarum polycephalum

“…One would expect to find only a low percentage of such molecules in purified rDNA since (a) it takes about 8 h (an entire mitotic cycle minus the first hour) to double the population of about 300 ribosomal genes [2, 13,23], or 150 rDNA molecules, and (b) a single molecule probably replicates in less than 30 min. The latter assumption is based on the known rate of fork displacement in higher eucaryotes, about 2 x lo6 g mol-l min-' [14] and on our preliminary experiments with density labeling, which showed that intact rDNA of hybrid density appeared in less than 30 min after placing a G-2 plasmodium in medium with BrdUrd.…”

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

“…polycephulum hybridises with the cytoplasmic rRNAs [33,14]; the figure is independent of ploidy [14] and there are about 300 genes for rRNA per haploid genome. The genes have been identified as a component of a 'heavy' nuclear satellite DNA, the replication of which is unusual in that it occurs throughout the mitotic cycle [32,33,38], with the possible exception of the first hour after mitosis [32].…”

“…The frozen preparations, which were stored in liquid N2 until required, were homogenised in deproteinisation medium and RNA isolated as described previously [14]. Fractions containing the 19-S and 2 6 4 rRNAs from sucrose density gradients were combined and the concentration of rRNA determined by measuring the absorbance at 258 nm (A;:,, = 221).…”

Ribosomal RNA Genes in Kluyveromyces marxianus

“…The difference in chromosome number between S. cerevisiae (17) and K. marxianus (9) should not account for this difference, since Neurospora crassa with 7 chromosomes has 100 cistrons per nucleus (Chattopadhyay et al, 1972). Furthermore, an increase in the level of ploidy does not lead to any increase in the number of cistrons per nucleus in Physarum polycephalum (Hall et al, 1975).…”