Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component.

Ribosomes from higher-plant mitochondria contain 5S rRNA, in contrast with the mitochondrial ribosomes of animals and fungi, in which such a component has not been detected. In common with the ribosomes of prokaryotes and chloroplasts, higher-plant mitochondrial ribosomes do not appear to contain an RNA equivalent to the 5.8 S rRNA that is found in eukaryoytes hydrogen-bonded to the largest of the cytoplasmic rRNA species.     

Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts of Euglena gracilis

1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.     

Mitochondrial and cytoplasmic ribosomes. Distinguishing characteristics and a requirement for the homologous ribosomal saltextractable fraction for protein synthesis

1. Mitochondrial and cytoplasmic ribosomes of Euglena gracilis differ in their total RNA and protein content. 2. Mitochondrial ribosomes dissociate to subunits at higher Mg(2+) concentrations than do cytoplasmic ribosomes. 3. A separable 5S RNA is obtained from cytoplasmic and chloroplast ribosomes, but not from mitochondrial ribosomes. 4. For protein-synthesizing activity with a natural mRNA, mitochondrial ribosomes use tRNA from any cell compartment and are partly active with supernatant enzymes from cytoplasm. Cytoplasmic ribosomes are partly active with enzymes and tRNA from mitochondria or chloroplasts. 5. Both mitochondrial and cytoplasmic ribosomes show high specificity for the homologous salt-extractable ribosomal fraction for protein-synthesizing activity.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells.

The production of cytoplasmic and nucleolar rRNA species was examined in HeLa cells infected with high multiplicities of adenovirus type 5. Both 28S and 18S rRNA newly synthesized in infected cells ceased to enter the cytoplasm as reported previously (N. Ledinko, Virology 49: 79-89, 1972; H. J. Raskas, D. C. Thomas, and M. Green, Virology 40: 893-902, 1970). However, the effects on 28S cytoplasmic rRNA were observed considerably earlier in the infectious cycle than those on 18S rRNA. The inhibition of cellular protein synthesis and of the appearance in the cytoplasm of labeled cellular mRNA sequences (G. A. Beltz and S. J. Flint, J. Mol. Biol. 131: 353-373, 1979) were also monitored in infected cultures. During the later periods of an infectious cycle, from 18 h after infection, nucleolar rRNA synthesis and processing and exit of 18S rRNA from the nucleus were inhibited, probably reflecting the failure of infected cells to synthesize normal quantities of ribosomal proteins. The earliest responses of cellular RNA metabolism to adenovirus infection were, however, the rapid and apparently coordinate reductions in the levels of newly synthesized 28S rRNA and cellular mRNA sequences entering the cytoplasm.     

Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture.

Growing and confluent cultures of mouse fibroblasts were labeled with ³H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing, and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.     

Ribosomal-type ribonucleic acid from rodent mitochondria

1. Highly purified mitochondria containing 3.0mug of RNA/mg of mitochondrial protein were prepared from rat liver by differential centrifugation. 2. RNA, labelled with [(32)P]P(i) or [(3)H]orotate, was isolated from these mitochondria by a phenol extraction method. The RNA sedimented at 15S and 13S on sucrose density gradients. Its nucleotide composition was 23% uridylate, 30% adenylate, 22% guanylate and 25% cytidylate. 3. RNA from mouse L cells was labelled with [(3)H]-uridine in the presence of 0.1mug of actinomycin D/ml to suppress the synthesis of cytoplasmic rRNA. The RNA isolated from crude L-cell mitochondria by a cold-phenol-sodium dodecyl sulphate method had components sedimenting at 15S and 12.5S. These components had an electrophoretic mobility on agarose-acrylamide gels of 21 and 12S(E) compared with 28 and 18S(E) for cytoplasmic rRNA. The nucleotide composition was 26% uridylate, 34% adenylate, 18% guanylate and 22% cytidylate. 4. RNA extracted from crude L-cell mitochondria by a hotphenol-sodium dodecyl sulphate method had an additional component sedimenting at 21S and having an electrophoretic mobility of 18S(E). It was probably DNA because of its sensitivity to deoxyribonuclease and its insensitivity to ribonuclease and alkali. It was present in nuclear fragments contaminating the crude mitochondrial fraction and could be removed by deoxyribonuclease or isopycnic-gradient centrifugation.     

Human glutamate tRNA forms stable hybrids in vitro with 28S ribosomal RNA.

A human glutamate tRNA has been shown to form stable hybrids with 28S ribosomal RNA. This tRNA was purified from HeLa cell cytoplasmic RNA by RNA-RNA solution hybridization followed by the isolation of tRNA-28S rRNA complexes by hybridization-selection with ribosomal DNA or by recovery of the 28S peak from formamide-sucrose gradients. The single hybridizing tRNA species was identified as tRNAGluCUC by sequencing: pU-C-C-C-U-G-G-U-G-m2G-U-C-phi-A-G-U-G-G-D-phi-A-G-G-A-U-U- C-G-G-C-G-C-U-C-U-C-A-C-C-G-C-G-G-C-m5C-m5C-G-G-G-Tm-phi-C-G-A- U-U-C-C-C-G-G-U-C-A-G-G-G-A-A-C-C-AOH. Computer analysis located a nucleotide sequence near the middle of human 28S rRNA which is complementary to 15-26 nucleotides between residues 20 and 50 of this tRNA. An interaction between this tRNA and 28S rRNA suggests that tRNAGluCUC may have functions in the cell in addition to translation.     

CHARACTERIZATION OF RAT BRAIN RIBONUCLEIC ACIDS BY AGAR GEL ELECTROPHORESIS

Abstract— —The characteristics of total and rapidly-labelled RNAs of rat brain were studied by agar gel electrophoresis. The bulk (more than 90 per cent) of total, nuclear and cytoplasmic brain RNA was represented by the 28 S, 18 S and 4 S RNA components. The 28 S/18 S RNA mass ratio in cytoplasmic RNA was 2·55. Lower values for this ratio were obtained with total and nuclear RNAs. Five minor RNA components were detected in total brain RNA with mobilities in agar gel corresponding to 24 S, 22 S, 14 S, 9 S and 6 S. Two broad rapidly labelled RNA components were detected in total and nuclear (but not in cytoplasmic) brain RNA with mobilities corresponding to about 45 S and 31 S. These fractions were of nuclear origin and resembled ribosomal precursor RNAs of other animal tissues. In cytoplasmic RNA the radioactivity and ultraviolet profiles coincided at all labelling times down to 1 hr. The G + C/A + U ratio of brain RNA was 1·50 for total RNA, 1·39 for nuclear RNA and 1·59 for cytoplasmic RNA. The G + C/A + U ratio of 1 hr-labelled total brain RNA (determined by ³²P-distribution) was 0·94. This ratio rose to 1·31 at 24 hr labelling. The possible significance of these results for the elucidation of ribosomal and messenger RNA metabolism in brain is discussed.     

In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.     

Lipid composition of plant mitochondria and of chloroplasts

The mitochondrial lipids from avocado fruit, cauliflower buds, and potato tubers, and the lipids of chloroplasts isolated from avocado fruit and from cauliflower leaves were identified and the concentrations were determined. The lipid composition was compared with that of beef heart mitochondria. Phospholipids constituted 50-56% of total lipids in plant mitochondria while this fraction made up 90% of the lipids in beef heart mitochondria. In both cases the chief phospholipids were phosphatidylcholine and phosphatidylethanolamine. A characteristic feature of plant mitochondria was the presence of monogalactosyl- and digalactosyldiglyceride and of sulfolipid. Potato mitochondria differed from the particles of other species investigated by their higher content of galactolipids, sterol glycosides, and carotenoids and lower content of phospholipids and of total lipids in the lipidprotein complex. The galactolipid content was markedly higher in chloroplasts from all sources than in mitochondria. The spectrum of lipids in the phospholipid fraction differed more strikingly between chloroplasts of the leaf and the mitochondria of the bud of cauliflower than between the two organelles of the avocado mesocarp. The fatty acid distribution of individual lipids and of classes of lipids was also more similar in the two organelles of the fruit tissue than in the cauliflower material.     

Phenol Extraction Op 28 S Ribosomal RNA Prom Rat Liver Cytoplasm

A simple and reproducible phenol method for the isolation of 28 S ribosomal RNA from rat liver cytoplasm, free from poly(A)-RNA is described. The procedure is based on the observation that at lower pH of the homogenate (pH 5.5) 28 S ribosomal RNA is extracted, while 18 S ribosomal RNA remains in the interphase layer.

Isolation of pure 28 S or 18 S ribosomal RNA in preparative amounts requires density gradient cen-trifugation or preparative gel electrophoresis. In this communication a rapid and reproducible method for the isolation of 28 S ribosomal RNA is proposed.