Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Vorstufen der ribosomalen RNA in frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary Six high molecular weight, rapidly labelled RNA species were detected in freely suspended callus cells of Petroselinum sativum by means of isotope labelling and electrophoretic separation in agarose-polyacrylamide gels. On the basis of their migration in the latter the RNA species were calculated to have the following molecular weights: 2.9×10⁶, 2,4×10⁶, 1.9×10⁶, 1.4×10⁶, 1.0×10⁶ and 0.75×10⁶ daltons. Thus they can clearly be distinguished from the two ribosomal RNA species (1.3×10⁶ and 0.7×10⁶ daltons). During incubation of the cells with [³H]methyl-methionine as a methyl donator all six components incorporated radioactivity rapidly. With [³H]nucleosides or [³H]orotic acid as precursors the 2.9×10⁶ and the 2.4×10⁶ daltons RNA were labelled within 10 min, while the other high molecular weight species appeared after about 20 min of labelling.Prolongation to 45–120 min resulted in accumulation of radioactivity preferentially in the 1.4×10⁶ and 0.75×10⁶ daltons RNA and in the ribosomal RNA species. The results of cell fractionation experiments provide evidence that these rapidly labelled high molecular weight RNA species are synthesized in the cell nucleus. The kinetics of their synthesis together with the other data obtained strongly support the suggestion that these RNA species function as precursors in the processing of ribosomal RNA. The possible mechanism of this process is discussed.

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Verwendete Abkürzungen EDTA Äthylendiamintetraessigsäure - DNase Desoxyribonuclease - Imp./min epm - MAK methyliertes Albumin an Kieselgur - POPOP 1,4- bis (4-Methyl-5-Phenyloxazol)-Benzol - PPO 2,5-Diphenyloxazol - RNase Ribonuclease - S Sedimentationskoeffizient in Svedberg-Einheiten - SDS Natriumdodecylsulfat - TPE Tris-Phosphat-EDTA-Puffer - Tris Tris-(hydroxymethyl)-aminomethan - Upm rpm     

On the size relationship between nuclear and cytoplasmic RNA in sea urchin embryos

The approximate sizes of heterogeneous nuclear (HnRNA) and cytoplasmic RNA of sea urchin embryos were determined by DMSO density gradient centrifugation and acrylamide-formamide gel electrophoresis. The data suggest that the sizes of these molecules are smaller than those estimated under nondenaturing conditions. The size of most of the nuclear RNA ranges from 0.5 to 3 × 10⁶ daltons, while that of the cytoplasmic RNA ranges from 0.1 to 2 × 10⁶ daltons. Both nuclear and cytoplasmic RNA of sea urchin embryos may have a minor fraction (5–10%) of very large species with molecular weights up to 4 to 5 × 10⁶ daltons.The idea that the size of HnRNA may be larger in organisms higher on the evolutionary scale is discussed.     

An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri

The arrangement of the genes and spacers has been analyzed in ribosomal DNA of Xenopus laevis and Xenopus mulleri by heteroduplex mapping and visualization of ribosomal RNA-DNA hybrids. Heterologous reassoeiated molecules show a characteristic pattern in which two perfectly duplexed regions, whose lengths are those predicted by the known lengths of the 18 S and 28 S genes, are separated by a small substitution loop of about 0.23 × 10⁶ daltons and a large region of partial homology which averages 3.24 × 10⁶ daltons. These mismatched regions are entirely consistent with the known sequence divergence previously described (Brown et al., 1972) for the transcribed and non-transcribed spacer regions of the two rDNAs, respectively. Hybrids of X. laevis rDNA with 18 S and 28 S rRNA contain two duplex regions of the expected lengths for the 18 S and 28 S genes separated by 0.49 × 10⁶ daltons of single-strand DNA. This latter value is the length of the transcribed spacer region between the 18 S and 28 S RNAs that has been measured within the 40 S RNA precursor molecule by secondary structure mapping (Wellauer &; Dawid, personal communication). There is also a longer single-strand region separating one 18 S + 28 S gene set from the next; this is considered to be mainly non-transcribed spacer.We conclude that the 18 S and 28 S genes are separated by about 0.5 × 10⁶ daltons of DNA of which about half is homologous in the two Xenopus species. This region is part of the transcribed spacer. In addition, the longer non-transcribed spacer can be seen to have some homology between the two species; the location of this homology is fairly reproducible between molecules and has been carefully documented by contour length measurements.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Removal of pectins from methylated rRNA and precursors by LiCl

The methyl group of radioactive methylmethionine is incorporated preferentially into pectin, a methylated polysaccharide, which massively contaminates RNA preparations. This makes it difficult to discern the methylated RNA species of growing tissues of higher plants. A method for the extraction of pectic substances from RNA preparations of plants is described. Ethanol precipitates containing RNA are suspended in 2m lithium chloride (LiCl) and the pectin is removed with the supernatant after centrifugation. LiCl purification of RNA from Vicia faba root meristems allows the direct identification on polyacrylamide gels of methylated RNA species labeled with [³H]methyl methionine. Presumpive precursors of rRNA which fall into the range of apparent molecular weights of 2.7–2.9, 2.2–2.4, 1.4–1.5, and 0.72–0.75 × 10⁶ are shown to be methylated, in addition to the methylation of the fully processed rRNAs of MW 1.3 and 0.7 × 10⁶.     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

Discontinuous DNA replication in developing sea urchin embryos.

Embryos of the sea urchin, Hemicentrotus pulcherrimus, growing synchronously and entering the third “S” phase at 120 min after fertilization, were pulse-labeled with [³H]thymidine for various periods ranging from 15 sec to 20 min, and the size of nascent DNA was analyzed by centrifugation in an alkaline sucrose gradient. It was found that pulse-labeling for 15 sec gave rise to a sedimentation profile with a major radioactivity peak at the position corresponding to a molecular weight of 4 × 10⁴ daltons. One-minute of labeling, however, gave a major radioactive band around the position corresponding to 1.4 × 10⁶ daltons. Upon increasing the labeling time, the radioactivity peaks or bands shifted toward the increasing molecular weights. Finally, most of the radioactive DNA was found to sediment at the bottom when the embryos were exposed to [³H]thymidine for 15 min or longer. The time span of the S phase in the cleavage embryos was about 15 min. The results of pulse and chase experiments also supported the discontinuous mechanism of DNA replication in the cleavage embryos.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

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.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic Acid

Mitochondria were prepared from etiolated maize shoots (Zea mays L. var. McNair 508) by homogenization followed by differential centrifugation and equilibrium banding in discontinuous sucrose or Renografin-sucrose gradients. Mitochondria prepared by sucrose banding showed better physiological integrity than those prepared by renografin-sucrose banding, although both procedures yielded mitochondria that showed respiratory control and coupling of oxidation to phosphorylation of ADP. Mitochondria prepared by Renografin-sucrose banding were free of dectectable cytoplasmic ribosomal RNA, while sucrose banding resulted in a low level of contamination. Ribosomes isolated from mitochondria sedimented at about 78S, with subunits sedimenting at 60 and 44S. Using Escherichia coli ribosomal RNA as internal standards, the molecular weights of mitochondrial ribosomal RNAs were found to be 0.74 to 0.75 and 1.26 × 10⁶ daltons by polyacrylamide gel electrophoresis, before or after denaturation in formaldehyde. Cytoplasmic ribosomal RNA molecular weights were 0.70 and 1.26 × 16⁶ before denaturation, and 0.68 and 1.5 × 10⁶ after denaturation, suggesting an unusual reaction of the heavy ribosomal RNA to formaldehyde.     

The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses

Ribonucleic acids extracted from polyribosomes of cells replicating murine sarcoma-leukemia viruses (M-MSV(MLV)) were resolved by electrophoresis on 2.5% polyacrylamide gels. Virus-specific RNA was detected by hybridization of RNA in the gel fractions with the ³H-DNA product of the viral RNA-directed DNA polymerase. The postmicrosomal supernatant and the free polyribosomes contained one peak of virus-specific RNA with a molecular weight of about 2.9 × 10⁶ (35S). In contrast, the microsomes and the membrane-bound polyribosomes contained two peaks of virus-specific RNA in approximately equal amounts with molecular weights of 2.9 × 10⁶ (35S) and 1.5 × 10⁶ (approximately 20S). The high molecular weight viral RNA species might serve as polycistronic mRNA for the synthesis of large polypeptides that are cleaved to form the smaller viral proteins.