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PubMed1977

Visualization of ribosomal ribonucleic acid synthesis in a ribonuclease III-Deficient strain of Escherichia coli.

Transmission electron microscopy was used to examine active ribosomal ribonucleic acid (rRNA) genes in two strains of Escherichia coli: N2077, deficient in the enzyme responsible for proper cleavage of the 16S sequence from the elongating nascent rRNA transcript; and N2076, functional in ribonuclease (RNase) III activity, yet otherwise isogenic to N2077. In the strain with wild-type RNase III, double gradients corresponding to a pattern of 16S-cleavage-23S transcription were observed. However, the RNase III-deficient strain exhibited a single ribosomal gradient of approximately the same length as the combined 16S-23S gradients of the wild-type strain. When the rRNA genes were somewhat loosely packed with RNA polymerases, a few of the nascent chains in the ribosomal matrixes of the RNase III-deficient strain were cleaved, but most appeared to be unprocessed. The completed, uncleaved transcripts originating from these gradients are believed to be 30S rRNA molecules recently characterized by biochemical probes.

DNA-Directed RNA Polymerases↗
PubMed1981

Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration.

The thiomethylation of Bacillus subtilis tyrosine transfer ribonucleic acid (tRNATyr) (i6A) has been shown to occur during the slowing-down of growth. The extent of this modification in stationary-phase cells grown in defined medium has been determined in parallel with the sporulation frequency. We observed that the presence of phosphate repressed sporulation and also inhibited the thiomethylation of tRNATyr (i6A) of B. subtilis W168. These effects were partially eliminated by decreasing the glucose concentration until it was growth limiting. In the case of strain W23S, in which sporulation is insensitive to glucose repression, sporulation and tRNATyr thiomethylation were not inhibited by nonlimiting concentrations of phosphate. These results suggest that both sporulation and tRNATyr hyper-modification share some common regulatory process.

Bacillus subtilis↗
PubMed2000

G8363A mutation in the mitochondrial DNA transfer ribonucleic acidLys gene: another cause of Leigh syndrome.

We identified a G-->A transition at nt-8363 in the mitochondrial DNA transfer ribonucleic acidLys gene in blood and muscle from a 13-month-old girl who had clinical and neuroradiologic evidence of Leigh syndrome and died at age 27 months. The mutation was less abundant in the same tissues from the patient's mother, who developed myoclonus epilepsy with ragged red fibers (MERRF) in her late 20s. In both mother and daughter, muscle histochemistry showed ragged red and cytochrome c oxidase-negative fibers and biochemical analysis showed partial defects of multiple respiratory-chain enzymes. A maternal half-sister of the proband had died at 2.5 years of age from neuropathologically proven Leigh syndrome. The G8363A mutation, which previously had been associated with cardiomyopathy and hearing loss, MERRF, and multiple lipomas, also should be included in the differential diagnosis of maternally inherited Leigh syndrome.

Adult↗
PubMed2000

Distribution and abundance of messenger ribonucleic acid for growth hormone receptor isoforms in human tissues.

Two alternatively spliced exon 9 variants of human GH receptor (GHR) messenger ribonucleic acid (mRNA), GHR-(1-279) and GHR(1-277), were recently identified in liver. They encode receptor proteins lacking most of the intracellular domain and inhibit GH action in a dominant negative manner. Little is known about tissue distribution and abundance of these GHR isoforms. We have developed quantitative RT-PCR assays specific for the full-length and truncated GHRs and investigated their expression in various human tissues and cell lines. The mRNA of full-length GHR and GHR-(1-279) were readily detectable in all tissues investigated, with liver, fat, muscle, and kidney showing high levels of expression. These two receptor isoforms were also detected in a range of human cell lines, with strongest expression in IM9, a lymphoblastoid cell line. In contrast, GHR-(1277) message was expressed at low levels in liver, fat, muscle, kidney, and prostate and in trace amount in IM9 cells. Full-length GHR was the most abundant isoform, accounting for over 90% of total receptor transcripts in liver, fat, and muscle for quantitative RT-PCR. However, liver had 2- to 4-fold more full-length receptor mRNA and 16- to 40-fold more GHR-(1-277) mRNA than fat and muscle, whereas the mRNA levels of GHR-(1-279) were similar in the three tissues. GHR-(1-279) constituted less than 4% in liver and 7-10% in fat and muscle. GHR-(1-277) accounted for 0.5% of total GHR transcripts in liver and less than 0.1% in the other two tissues. These data suggest that the absolute and relative abundance of mRNA of the three GHR isoforms may be tissue specific. The regulation of expression of exon 9 alternatively spliced GHR variants may provide a potential mechanism for modulation of GH sensitivity at the tissue level.

Adipose Tissue↗
PubMed2004

Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants.

The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by beta-naphthoflavone (betaNF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription-polymerase chain reaction. The concentration-dependent induction of CYP1A mRNA in the liver was observed after exposure to betaNF at nominal concentrations of 20, 100, and 500 microg/L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 microg/L of betaNF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to betaNF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the betaNF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals.

Amino Acid Sequence↗
PubMed1980

Characterization of the viral ribonucleic acids and structural polypeptides of Anopheles A, Bunyamwera, Group C, California, Capim, Guama, Patois, and Simbu bunyaviruses.

Analyses of the viral ribonucleic acids and structural polypeptides of 17-22 of the 119 accepted or proposed members of the Bunyavirus genus of arboviruses (family Bunyaviridae), have shown that from the standpoint of their structural components these viruses are highly comparable to each other. The average molecular weights for the three viral RNA species (L, large, M, medium, S, small) of 17 bunyaviruses were 2.93 X 10(6) (L, range 2.7-3.1 X 10(6)), 2.0 X 10(6) (M, range 1.8-2.3 X 10(6)), and 0.435 X 10(6) (Sm range 0.28-0.50 X 10(6)). The average molecular weights of the three major virion polypeptides (glycoproteins G1 and G2, and nucleocapsid protein, N) of 22 bunyaviruses were 115 X 10(3) (G1, range 108-120 X 10(3)), 37 X 10(3) (G2, range 20-41 X 10(3)) and 22 X 10(3) (N, range 19-25 X 10(3)). These results indicate that the structural components of bunyaviruses are different from those reported for Phlebotomus fever, Uukuniemi, and Crimean-Congo hemorrhagic fever, and other members of the Bunyaviridae family that are not currently assigned to a genus.

Animals↗
PubMed2003

Stable isotope labeling for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay analysis of ribonucleic acids.

Matrix-assisted laser desorption/ionization mass spectrometry is a powerful analytical tool for the structural characterization of oligonucleotides and nucleic acids. Here we report the application of stable isotope labeling for the simplified characterization of ribonucleic acids (RNAs). An (18)O label is incorporated at the 3'-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. As implemented, a buffer solution containing a 50 : 50 mixture of H(2)O and (18)O-labeled H(2)O is used during endonuclease digestion. Upon digestion, characteristic doublets representative of the isotopic distribution of oxygen are noted for those products that contain 3'-phosphate groups. This approach is used to distinguish readily endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, RNase digestion products containing the characteristic isotopic doublet can be selected for further characterization by post-source decay (PSD) analysis. PSD products carrying the 3'-phosphate group will appear as a doublet, thereby simplifying fragment ion assignment.

Escherichia coli↗
PubMed1979

In vitro transcription by wheat germ ribonucleic acid polymerase II: effects of heparin and role of template integrity.

Double-stranded deoxyribonucleic acid (DNA) from bacteriophage lambda is a good template for wheat germ DNA-dependent ribonucleic acid (RNA) polymerase II. We delineated conditions for obtaining maximum polymerase activity using as template both the relatively intact DNA extracted from the the lambda phage and DNA into which single-strand nicks have been introduced by deoxyribonuclease (DNase) I digestion. The deliberate introduction of nicks produces a modest increase in transcription. The NaCl and MgCl2 optima are broader with the nicked template, so that higher concentrations of these salts are needed before polymerase activity begins to decline. Heparin inhibits initiation but not elongation by wheat germ polymerase. Polymerase can be protected against heparin inhibition by forming binary complexes with the template. The formation of these complexes is reduced at low temperature. The complexes, once formed, decay in the presence of heparin with a half-life of 10--20 min. The number of complexes is highly dependent on the degree of nicking of the template, suggesting that single-strand nicks are the predominant type of site where these heparin-resistant complexes are formed. Our data do not allow us to decide whether or not the presence of nicks plays as decisive a role in the absence of heparin.

DNA-Directed RNA Polymerases↗
PubMed1967

Regeneration of ribosomes and ribosomal ribonucleic acid during repair of thermal injury to Staphylococcus.

Heating Staphylococcus aureus MF31 at 55 C for 15 min renders the organisms unable to reproduce on agar containing 7.5% NaCl (1). The heated organisms exhibited an extended lag period during which the organisms regained their ability to grow on the 7.5% NaCl-agar. Inhibitor and antibiotic data indicated that protein synthesis is not involved in this recovery process but nucleic acid synthesis is suggested (3). The data presented here further substantiate the noninvolvement of protein synthesis during recovery and further demonstrate the site of the thermally induced nucleic acid lesion. Methylated albumin kieselguhr column analysis showed the lesion site to be the ribosomal ribonucleic acid (rRNA). The rRNA is resynthesized during the extended lag period. Sucrose gradient analysis demonstrated that a ribosomal peak was undetectable subsequent to the thermal treatment, but this peak was regenerated during the recovery period.

Bacterial Proteins↗
PubMed1969

Properties of adenovirus messenger ribonucleic Acid synthesized in vitro.

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 mug of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 mug of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).

Journal Article↗
PubMed1989

Electron microscopy of ribonucleic acid in nuclear particulate aggregates of hepatitis D using nuclease-gold complexes.

The ultrastructural localization of hepatitis delta antigen (HDAg) and ribonucleic acid (RNA) was investigated by immunoperoxidase electron microscopy and by enzyme electron microscopy of RNase-gold complexes on liver biopsies from seven patients with hepatitis D. HDAg was localized mainly in the nucleus and sometimes in the cytoplasm of hepatocytes. Ultrastructurally, intranuclear HDAg was found on nuclear particulate structures measuring 20 to 30 nm in diameter. Intranuclear RNA visualized with gold particles was found in high amounts in the nucleolus, to a small extent in the chromatin area, and also on nuclear particulate structures. These findings suggest that intranuclear aggregates of irregular granular particulate structures in hepatitis D are the internal component of hepatitis delta virus (HDV) particles in blood.

Antigens, Viral↗
PubMed1988

Accumulation of abundant messenger ribonucleic acids during postnatal development of mouse small intestine.

To describe the differentiation of the small bowel at the molecular level, intestinal messenger ribonucleic acids (mRNAs) from mice at different stages of fetal and postnatal development were investigated. On the basis of cell-free translation and complementary deoxyribonucleic acid cloning experiments, abundant mRNAs coding for small polypeptides of 6-12 kilodaltons (low-molecular-weight mRNAs) were found in adult small intestine but not in the fetal gut. These developmentally regulated low-molecular-weight mRNAs are uniquely abundant in jejunum and ileum of adult mice, but they are absent or occur only at low levels in the duodenum, colon, stomach, and all other mouse organs examined. Low-molecular-weight mRNAs begin accumulating in the small bowel at approximately 3 wk of age, coinciding with weaning and with profound changes in intestinal differentiation. One complementary deoxyribonucleic acid clone of a low-molecular-weight mRNA (asb4/134) is specific to the distal small bowel, specifically accumulates at weaning, and hybridizes to RNA from mouse testis and brain at approximately 2%-5% of the intestinal level. Low-molecular-weight mRNA sequences may provide important markers of intestinal differentiation at the genetic level, leading to a better understanding of the factors that contribute to its postnatal maturation.

Animals↗
PubMed1981

Processing of the ribonucleic acid in the large ribosomal subunits of Urechis caupo.

Ribosomal subunits were isolated from eggs or embryos of Urechis caupo, and the ribonucleic acid (RNA) was characterized by electrophoresis under denaturing conditions. The small ribosomal subunit contains a single 17S RNA sequence with a molecular weight of 6.20 X 10(5). The large ribosomal subunit contains four polynucleotide sequences. The 5S RNA has a molecular weight of 4.09 X 10(4). The 26S RNA complex isolated under nondenaturing conditions dissociates in the presence of formamide to yield a 5.8S RNA, molecular weight 5.46 X 10(4), and two approximately 17S and 17.5S RNA sequences with molecular weights of 6.04 X 10(5) and 6.61 X 10(5). The 17S and 17.5S RNAs of the large ribosomal subunits are formed in vivo from a 26S RNA precursor after assembly of the large ribosomal subunit. Large ribosomal subunits are transferred from the nucleus to the cytoplasm with the 26S RNA precursor intact. The hidden break to form the 17S and 17.5S RNAs is introduced in the cytoplasm. No intact 26S RNA could be detected in polysomes; this indicates that the conversion of the 26S RNA to the 17S and 17.5S RNAs may be required to produce large ribosomal subunits capable of participating in protein synthesis.

Animals↗
PubMed1979

Propagation of ribonucleic acid coliphages in gnotobiotic mice.

To clarify the propagation cycle of bacteriophages in their natural habitats, we tested whether animals could support ribonucleic acid (RNA) phage propagation in their intestines, using germfree mice as the test animal. Propagation of four different antigenic types of RNA phages was tested. No detectable propagation or colonization of RNA phages was observed either in germfree mice or in gnotobiotic mice infected with the F- strain of Escherichia coli. Propagation or colonization was observed when RNA phages were orally introduced into gnotobiotic mice harboring the F+ or F' strain of E. coli. These results were consistent with data for in vitro propagation experiments. Fecal titers of phages were monitored over 24 to 98 days and were found to vary from 10(5) to 10(11) plaque-forming units per g of feces. Streptomycin administration gradually led to the disappearance of bacteria and, concomitantly, the RNA phages. Phages recovered from gnotobiotic mice feces included some of novel antigenic types. The bacterial isolates recovered from gnotobiotic mice harboring F+ bacteria included the original F+ strain, strains which had become F-, and some which had become inefficient hosts for the propagation of RNA phages.

Animals↗
PubMed1972

Factors affecting virulence of Shigella flexneri: strain with relaxed control of ribonucleic acid synthesis.

An Escherichia coli-Shigella flexneri hybrid strain which produces an abortive, nonfatal experimental dysentery has inherited relaxed control of ribonucleic acid synthesis from its E. coli parent. This property, coupled with a partial methionine requirement, has been used to explain its long lag phase in vitro, relative to its Shigella parent. It was suggested that such characteristics may contribute to the hybrid's poor survival in the gut resulting in the abortive infection.

Alleles↗
PubMed1973

Increased toxicity of double-stranded ribonucleic acid in virus-infected animals.

Virus-infected mice were significantly more susceptible to the toxic effects of double-stranded ribonucleic acid (RNA) than uninfected mice. A dramatic increase in mortality was observed after injection of either synthetic (polyriboinosinic.polyribocytidylic acid) or natural (mycophage) double-stranded RNA in mice infected with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). With the exception of endotoxin, interferon inducers other than double-stranded RNA, such as tilorone-hydrochloride and chlorite-oxidized oxyamylose, did not show this increased toxicity in virus-infected animals. The increased susceptibility of virus-infected animals to the toxic effects of double-stranded RNA appears to be related to the levels of interferon induced by the virus infection, either systemically, in the blood stream (after inoculation of NDV), or locally, in the brain (after infection with VSV).

Animals↗
PubMed1971

Inhibition of arginyl-transfer ribonucleic acid synthetase activity of Escherichia coli by arginine biosynthetic precursors.

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.

Amino Acyl-tRNA Synthetases↗