Alteration in the processing of ribosomal ribonucleic acid inSaccharomyces cerevisiae caused by ethidium bromide

Ethidium bromide in a concentration of 200 μg/ml causes a full inhibition of RNA synthesis in aSaccharomyces cerevisiae ρ° strain, while protein synthesis continues at a reduced rate. Under these conditions, processing of rRNA is slowed down and part of the 37S rRNA precursor molecules are cleaved to a 32S RNA fraction (molecular weight 2.15×10⁶). The 32S RNA accumulates in cells treated with ethidium bromide but cannot be processed to mature 25S and 18S rRNA and is degraded. The 32S RNA fraction also appears when processing of rRNA occurs in cells starved for required amino acids. The degradation of 37S precursor molecules through 32S RNA may be a regulatory mechanism of rRNA biosynthesis in yeast, which operates when excess rRNA must be wasted.     

Sequence Studies on Precursor 16S Ribosomal RNA of <Emphasis Type="Italic">Escherichia coli</Emphasis>

THE 16S and 23S rRNAs of E. coli are known to be synthesized through intermediates of marginally. higher S values (about 17S and 24S). Although various authors^1–3 have reported the presence of these precursors, no sequence studies have been presented. Nevertheless, there is strong but indirect evidence³ that the precursor 16S rRNA (p16) is about 200 residues longer than 16S rRNA (ml6). In contrast, the evidence suggests that the precursor 23S rRNA has few or no further residues.     

Separation of Bacterial Ribosomal Ribonucleic Acid from Its Macromolecular Precursors by Polyacrylamide Gel Electrophoresis

Electrophoresis on polyacrylamide gels was found to be a powerful technique for separating the mature from the precursor forms of bacterial ribosomal nucleic acid (rRNA). The separation of the 16S rRNA from its precursor was, for all practical purposes, complete; that of the 23S rRNA from its precursor was detectable but incomplete. When mature and precursor rRNA preparations were heated to randomize secondary structure, etc., and then cooled, it was found that electrophoretic mobility differences between mature forms of rRNA and their precursors persisted. This, in conjunction with the rather large electrophoretic mobility differences between mature and precursor forms, can be taken as strong evidence for a molecular weight difference between mature rRNA and its precursor forms of RNA. With the 16S rRNA, this difference could be as large as 130,000 daltons.     

Transcription and processing of rRNA in higher plant cells (Daucus carota,Petroselinum crispum,Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [³²P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 10⁶ daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2^? 3.4 × 10⁶ daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 10⁶ daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 10⁶ daltons.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.     

Processing pathway of Escherichia coli 16S precursor rRNA.

Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA.     

Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber (Cucumis sativus L.) from32P-labelled organic matter during mineralization in soil

An experiment was set up to investigate the role of arbuscular mycorrhiza (AM) in utilization of P from organic matter during mineralization in soil. Cucumber (Cucumis sativus L.) inoculated with one of two AM fungi or left uninoculated were grown for 30 days in cross-shaped PVC pots. One of two horizontal compartments contained 100 g soil (quartz sand: clay loam, 1:1) with 0.5 g ground clover leaves labelled with³²P. The labelled soil received microbial inoculum without AM fungi to ensure mineralization of the added organic matter. The labelling compartment was separated from a central root compartment by either 37 m or 700 m nylon mesh giving only hyphae or both roots and hyphae, respectively, access to the labelled soil. The recovery of³²P from the hyphal compartment was 5.5 and 8.6% for plants colonized withGlomus sp. andG. caledonium, respectively, but only 0.6 % for the non-mycorrhizal controls. Interfungal differences were not related to root colonization or hyphal length densities, which were lowest forG. caledonium. Both fungi depleted the labelled soil of NaHCO₃-extractable P and³²P compared to controls. A 15–25% recovery of³²P by roots was not enhanced in the presence of mycorrhizas, probably due to high root densities in the labelled soil. The experiment confirms that AM fungi differ in P uptake characteristics, and that mycorrhizal hyphae can intercept some P immobilization by other microorganisms and P-sorbing clay minerals.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.