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-
Cell division was synchronised in 4-litre batch cultures of Acer pseudoplatanus L. by starvation and regrowth. Up to five consecutive cell cycles were observed in each culture. Mitosis and cytokinesis were synchronised within 0.2 cell cycles. Accumulation of extractable DNA was discontinuous and separate from cytokinesis. Correction for the degree of synchrony in the population gave: G1=13-37 h, S=15 h, G2=14-19 h and M=0.9-1.3 h. Thymidine kinase activity and [(14)C]thymidine incorporation were highest during S-phase. A peak of activity of aspartate transcarbamoylase occurred during G2. Peaks in succinate dehydrogenase activity and respiration rate were observed at the initiation of DNA synthesis and just prior to mitosis. The activity of glucose-6-phosphate dehydrogenase doubled in one step during the cell cycle. Total RNA and protein accumulated continuously through the cell cycle; the final rate being twice that observed initially.
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...
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