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At least 1,423 records · Page 79Linked to original sources
PubMed1967

Heterogeneity of the stability of messenger ribonucleic acid in Salmonella typhimurium.

Cells of Salmonella typhimurium strain SL 282, deflagellated by mechanical shear, regenerated their flagella in the absence of tryptophan, an amino acid required for growth but not found in flagellin. Ribonucleic acid (RNA) synthesis was severely inhibited by tryptophan starvation. These findings suggested that the messenger RNA (mRNA) for flagellin might be stable. Actinomycin D was used to inhibit RNA synthesis in ethylenediaminetetraacetate-treated bacteria. The introduction of an F(lac) episome into strain SL 282 permitted the simultaneous study of the synthesis of flagellin, beta-galactosidase, and total protein. In the actinomycin-treated bacteria protein and beta-galactosidase syntheses were inhibited by 90%, whereas flagellin synthesis was unaffected. We conclude that the mRNA for flagellin synthesis is stable and that species of mRNA vary with respect to metabolic stability in S. typhimurium.

Bacterial Proteins↗
PubMed1975

Specificity of the stimulation of in vitro ribonucleic acid synthesis by guanosine 5'-diphosphate 3'-diphosphate.

The in vitro synthesis of ribonucleic acid (RNA) by S-30 extracts of Escherichia coli K-12 is stimulated from two-to fourfold by 0.16 mM to 0.32 mM guanosine 5'-diphosphate 3'-diphosphate (ppGpp) when either gammacI857St68h80 deoxyribonucleic acid (gammah80 DNA), gammah80dilv DNA or gammah80dlac DNA are employed as templates. Hybridization analysis of the 3H-RNA product transcribed from gammah80dilv DNA in the presence of ppGpp indicates that both bacteriophage- and bacterial-specific transcription is stimulated to an equivalent degree. In the absence of cyclic 3'-5'-adenosine monophosphate (cyclic AMP), correct lac-specifci RNA synthesis from gammah80dlac DNA is not stimulated by 0.32 mM ppGpp although total RNA synthesis is increased nearly twofold. In the presence of 0.5 mM cyclic AMP, correct lacspecific RNA synthesis is stimulated preferentially by ppGpp. These data suggest that ppGpp is capable of stimulating in vitro transcription in both a general and selective manner.

Cyclic AMP↗
PubMed1986

Incorporation of labeled ribonucleic acid precursors into maternal and fetal rat tissues during pregnancy.

Tritium-labeled ribonucleic acid precursors, including cytidine, uridine, and orotic acid, were injected into rats with dated pregnancies (14 to 21 days) and virgin rats. The acid-insoluble counts indicating incorporation into fetal and placental tissues showed that the highest incorporation occurred with cytidine, particularly earlier in pregnancy. In contrast, uridine demonstrated a minor degree of incorporation but displayed facile and enhanced transplacental passage with duration of pregnancy as represented by acid-soluble counts. Orotic acid was minimally used by both fetal and placental tissues. The incorporation of labeled precursors into maternal liver, heart, and kidney demonstrated varying responses during the course of pregnancy.

Animals↗
PubMed1984

Inhibition of host cellular ribonucleic acid synthesis by glycoprotein of mumps virus.

After infection with mumps virus, cellular ribonucleic acid synthesis of a murine lymphoma cell line, EL4, was appreciably depressed. The inactivation of viral infectivity by ultraviolet irradiation or the treatment of cells with mouse interferon did not abolish the inhibiting effect, suggesting that virus replication is not required for the depressed RNA synthesis. Envelope glycoproteins isolated from disrupted mumps virus caused inhibition of cellular RNA synthesis. The addition of low concentrations of specific antibody enhanced the inhibitory effect, probably through the formation of aggregates of glycoproteins. On the contrary, the glycoproteins showed no effect on RNA synthesis in the presence of cytochalasin D.

Animals↗
PubMed1981

Denaturation and renaturation of Penicillium chrysogenum mycophage double-stranded ribonucleic acid in tetraalkylammonium salt solutions.

The base composition dependence of double-stranded ribonucleic acid (RNA) melting was studied by observing the structure and widths of melting transitions for Penicillium chrysogenum mycophage RNA as well as differences in melting temperatures of two RNAs of different base composition. Double-stranded RNA melting is independent of base compositions in 3.5 M Et4NCl and 4.6 M Me4NCl, where the melting temperatures are 25 and 92 degrees C, respectively. Double-stranded RNA renaturation rate constants are reported in Et4NCl solutions. The nucleation rate constant is about 10 times lower than that for double-stranded deoxyribonucleic acid. Analyses of renaturation kinetics results lead to the conclusion that each of the three similar but separable RNA segments of Penicillium chrysogenum mycophage is unique.

Kinetics↗
PubMed1981

Association of poly(adenylate)-deficient messenger ribonucleic acid with membranes in mouse kidney.

To describe further the metabolism of messenger ribonucleic acid (mRNA) in mouse kidney, we examined newly synthesized mRNA deficient in poly(adenylate) [poly(A)]. Approximately 50% of renal polysomal mRNA that labeled selectively in the presence of the pyrimidine analogue 5-fluoroorotic acid lacks or is deficient in poly(A) as defined by its ability to bind to poly(A) affinity columns. Nearly one-half of this poly(A)-deficient mRNA is associated uniquely with a cellular membrane fraction detected by sedimentation of renal cytoplasm in sucrose density gradients containing EDTA and nonionic detergents. Poly(A+) mRNA and poly(A)-deficient mRNA [poly(A-) mRNA] have similar modal sedimentation coefficients (20-22 S) and similar cytoplasmic distribution. Although 95% of newly synthesized poly(A+) mRNA is released in 10 mM EDTA as 20-90 S ribonucleoproteins from polysomes greater than 80 S, only 55% of poly(A)-deficient mRNA is released under the same conditions. Poly(A)-deficient mRNA recovered from greater than 80 S ribonucleoproteins resistant to EDTA treatment lacks ribosomal RNA, is similar in size to poly(A+) mRNA, and is associated with membranous structures, since 70% of poly(A)-deficient mRNA in EDTA-resistant ribonucleoproteins is released into the 20-80 S region by solubilizing membranes with 1% Triton X-100. These membrane-associated renal poly(A-) mRNAs could have unique coding or regulatory functions.

Animals↗
PubMed1980

Interaction of Escherichia coli host factor protein with Q beta ribonucleic acid.

The affinity of Escherichia coli host factor protein for a variety of ribonucleic acids (RNAs) is compared in an equilibrium competition assay with (pA)15 or (pA)27 as the common probe. Of the homopolymers tested, only polyriboadenylate [poly(rA)] binds the protein with a high affinity. At low ionic strength (0.1 M NaCl), the binding to Q beta RNA is much stronger than to the oligoadenylates, but the situation is reversed upon fragmentation of the RNA with ribonuclease T1. Increasing the ionic strength results in a drastic reduction of the affinity of host factor for Q beta RNA over a relatively narrow salt range (0.1--0.3 M NaCl). Over the same range, added salt greatly reduces the tendency of host factor hexamers to aggregate. The tight binding of host factor to Q beta RNA is proposed to result from the binding of an aggregate, which can interact with several low affinity sites on the RNA simultaneously.

Binding Sites↗
PubMed1971

Chromatographic separation of mononucleotides derived from transfer ribonucleic acids.

1. A method is described for fractionating alkaline and enzymic hydrolysates of ribonucleic acids and oligonucleotides on DEAE-cellulose columns by using a constant-composition eluent. 2. The relative partition coefficients of the four major and several minor nucleotides present in nucleic acid digests are given. 3. The influence of pH and of molarity of the buffer on the separation is described. 4. Relations for the quantitative determination of the major nucleotides from their absorption zone areas on the chromatographic record are derived. 5. Hydrolysis of oligonucleotides with T2 ribonuclease yields solely the nucleoside 3'-monophosphates, thereby simplifying the subsequent chromatographic separation. 6. Examples of the qualitative and quantitative analysis of oligonucleotides from enzymic hydrolysates of RNA are given.

Chromatography, DEAE-Cellulose↗
PubMed1964

RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS.

The pattern of ribonucleic acid synthesis during germ cell development, from the stem cell to the mature spermatid, was studied in the mouse testis, by using uridine-H(3) or cytidine-H(3) labeling and autoradiography. Incorporation of tritiated precursors into the RNA occurs in spermatogonia, resting primary spermatocytes (RPS), throughout the second half of pachytene stage up to early diplotene, and in the Sertoli cells. Cells in leptotene, zygotene, and in the first half of pachytene stage do not synthesize RNA. No RNA synthesis was detected in meiotic stages later than diplotene, with the exception of a very low rate of incorporation in a fraction of secondary spermatocytes and very early spermatids. At long intervals after administration of the tracer, as labeled cells develop to more mature stages, late stages of spermatogenesis also become labeled. The last structures to become labeled are the residual bodies of Regaud. Thus, the RNA synthesized during the active meiotic stages is partially retained within the cell during further development. The rate of RNA synthesis declines gradually with the maturation from type A to intermediate to type B spermatogonia and to resting primary spermatocytes. "Dormant" type A spermatogonia synthesize little or no RNA. The incorporation of RNA precursors occurs exclusively within the nucleus: at later postinjection intervals the cytoplasm also becomes labeled. In spermatogonia all mitotic stages, except metaphase and anaphase, were shown to incorporate uridine-H(3). RNA synthesis is then a continuous process throughout the cell division cycle in spermatogonia (generation time about 30 hours), and stops only for a very short interval (1 hour) during metaphase and anaphase.

Animals↗
PubMed2007

Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.

HYPOTHESIS: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. BACKGROUND: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. METHODS: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. RESULTS: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. CONCLUSION: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Animals↗
PubMed1972

Optimization of the interferon assay using inhibition of Semliki forest virus-ribonucleic acid synthesis.

The method for assaying chicken interferon by its inhibition of viral ribonucleic acid (RNA) synthesis was optimized for the chicken embryo fibroblast-Semliki Forest virus (SFV) system, with respect to time, multiplicity of infection, and addition of actinomycin D and (3)H-uridine. Incorporation of (3)H into viral RNA is reproducible, and amounts to 10(4) to 2 x 10(4) counts per min per uninhibited culture per 1 muCi per 6 hr. The assay may be carried out in less than 1 day and is sensitive (0.05 units/ml) and exact (+/-20% of the mean titers on different days); it can be used for purification procedures as well.

Animals↗
PubMed1970

Immunogenicity of Ribonucleic Acid Preparations Obtained from Salmonella typhimurium.

Mice immunized with purified whole-cell ribonucleic acid (RNA), RNA from the bacterial "particulate" fraction, and ribosome-associated RNA obtained from Salmonella typhimurium were found to be resistant to subsequent challenge infection with virulent salmonellae. Chemically, the immunogenic nucleic acid fractions contained from 1 to 3% "contaminant" material defined (based on the mean of 19 different preparations) as protein (0.24%), deoxyribonucleic acid (0.43%), methyl pentose (0.64%), hexose (1.58%), and undefined carbohydrate (0.76%). Heptoses and lipoidal material were not detectable in any of the immunogenic preparations examined. Physically, the nucleic acid preparations, after analytical ultracentrifugation, exhibited three boundaries similar to those reported elsewhere in comparable systems: 4 to 5S, 16S, and 23S. An evaluation of the immunity induced by the ribosome-associated RNA established that the immune response was (i) comparable to that induced 15 days postimmunization with live salmonellae and by ribosomal vaccines, but greater at 30 days postimmunization than that in mice immunized with attenuated salmonellae; (ii) dependent on the quantity of immunogen administered; (iii) dependent on the size of the infective inocula; (iv) inhibited at 15 but not at 30 days postimmunization when the immunogenic nucleic acid preparations were incorporated into Freund's incomplete adjuvant, (v) reduced or lost by dialysis in relatively high or low immunizing doses, respectively; and (vi) unaffected by enzymatic treatment of the preparations with trypsin, deoxyribonuclease, Pronase plus pancreatic ribonuclease, or pancreatic ribonuclease alone. The possible mode of action of ribosome-associated RNA in inducing an immune response to subsequent challenge infection with the homologous organism is discussed.

Journal Article↗
PubMed1973

Ribonucleic acid and protein synthesis in guinea pig liver during Q fever.

Protein and ribonucleic acid (RNA) syntheses increase in the liver during Q fever, coincident with progressive increments in cortisol levels. Cell-free protein-synthesizing systems indicate that the pH 5 enzyme fraction was chiefly responsible for enhanced activity. Although the polysome profiles from normal and infected livers were similar, the number of liver ribosomes doubled during infection. There was a concomitant increase in orotate incorporation in the 28s, 18s, and 4s RNA species of infected liver.

Amino Acids↗
PubMed1969

Decay of normal and 5-fluorouracil-substituted messenger ribonucleic acid of alkaline phosphatase in Escherichia coli.

The decay of alkaline phosphatase messenger ribonucleic acid (mRNA) in Escherichia coli was studied in cells sensitized to actinomycin D by ethylenediaminetetra-acetate treatment. It was found that in the wild-type strain (K-10) as well as in a nonsense mutant (S26C200), which is phenotypically reversible by treatment with 5-fluorouracil, mRNA decays with a half-life of 2.0 to 2.5 min. Similarly, in a 3-min pulse of labeled 5-fluorouracil, 70% of the incorporated analogue is contained in the labile RNA fraction decomposing with a half-life of 1.8 min.

Alkaline Phosphatase↗
PubMed1972

Synthesis, no synthesis, or synthesis and degradation of ribosomal ribonucleic acid in various Escherichia coli strains starved for an amino acid.

A standard stringent strain of Escherichia coli makes little or no ribosomal ribonucleic acid (RNA) during starvation for an essential amino acid, whereas the isogenic relaxed strain makes both ribosomal and messenger RNA. A third class of strains was found which continues to make ribosomal RNA during starvation, but the RNA made is apparently unstable. There is little accumulation of RNA in the third class of strains, and few complete newly formed chains of (3)H-ribosomal RNA are observed in sedimentation analyses, even after long labeling times.

Amino Acids↗
PubMed1974

Absence of 3'-terminal residues from transfer ribonucleic acid of dormant spores of Bacillus megaterium.

Essentially all (>97%) of the transfer ribonucleic acid (tRNA) in log-phase and sporulating cells of Bacillus megaterium contains a complete 3'-cytidyl-cytidyl-adenosine terminus. However, about one-third of the tRNA in the dormant spore lacks the 3'-terminal adenosine 5'-monophosphate (AMP) residue, and some of the adjacent cytosine monophosphate residues are also missing. Examination of specific tRNAs indicated that those specific for isoleucine, leucine, and methionine are missing 30 to 40% of their terminal residue, whereas tRNAs specific for tyrosine lack 88% of the 3'-terminal AMP. Defective spore tRNA is not degraded during germination, but the missing residues are added back in the first minutes of the process. The enzyme catalyzing the addition reaction, tRNA nucleotidyltransferase, is present in the dormant spore at a level similar to that found in the vegetative cell.

Adenosine Monophosphate↗