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Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis.

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.

Adenosine↗

Age-related changes in function of transfer ribonucleic acid of rat livers.

Changes in transfer ribonucleic acids during aging could be caused by alterations in regulation or mutation and give rise to slower and less accurate protein synthesis. Rodent liver parenchymal cells, purified from disaggregated livers, do decrease in ability to incorporate labeled amino acids during aging. Moreover, old rodents have a rapidly degraded fraction of liver soluble RNA which is absent from middle-aged animals. In addition, tRNAs purified from old unfractionated liver cannot be acylated as well as from young. High speed supernatant tRNAs from old and young liver are quite similar in acylation capacity. Analysis indicates that a defective subfraction of tRNA may be bound to the ribosomal fraction of the liver cell. Some evidence indicates that base modification levels differ in young and old rodent liver. Shifts in the proportions of lysine and serine isoacceptors during aging are consistent with this idea. One isoacceptor change is an increase in tRNAlys4, which is correlated with cell division capacity in other systems.

Acylation↗

RIBONUCLEIC ACIDS OF BACILLUS SUBTILIS SPORES AND SPORULATING CELLS.

Doi, Roy H. (Syracuse University, Syracuse N.Y.), and Richard T. Igarashi. Ribonucleic acids of Bacillus subtilus spores and sporulating cells. J. Bacteriol. 87:323-328. 1964.-The ribonucleic acids (RNA) of Bacillus subtilis spores were analyzed for their size and base composition. Soluble and ribosomal RNA identical to those found in vegetative cells were present in spores. A base ratio difference was observed between the 16s and 23s ribosomal RNA. The 16s RNA had a higher cytidylate and lower adenylate base composition. The ratio of soluble of ribosomal RNA in spores was 30%, in contrast to 15% in vegetative cells, and may be related to their difference in biosynthetic activity. Although active synthesis of informational RNA occurred prior to endospore formation, no detectable level of informational RNA was found in spores. The informational RNA was shown to have the same base composition as that of B. subtilis deoxyribonucleic acid. The compositional difference between spore and vegetative cell RNA appeared to be strictly quantitative.

Bacillus↗

INTRACELLULAR FATE OF MENGO VIRUS RIBONUCLEIC ACID.

Homma, M. (The Wistar Institute, Philadelphia, Pa.), and A. F. Graham. Intracellular fate of Mengo virus ribonucleic acid. J. Bacteriol. 89:64-73. 1965.-P(32)-labeled, purified preparations of Mengo virus adsorbed rapidly and irreversibly to L cells maintained in suspension cultures. At intervals after adsorption of labeled virus, the total ribonucleic acid (RNA) of infected cells was extracted by a phenol technique. Infectivity titrations on this RNA showed that it retained its full biological activity during the early part of the eclipse period. Sucrose gradient sedimentation analyses showed also that this RNA lost none of its structural integrity throughout the eclipse period. No evidence was found for a double-stranded structure involving parental RNA. When cells infected with P(32)-labeled virus were broken open during the first 7 hr after infection, no more than 20% of the parental RNA could be digested with ribonuclease. Electron microscopy indicated that only one particle in five of the purified viral populations was infectious. It is suggested that only one of five adsorbed virus particles was uncoated and that its RNA remained in the cell in an undegraded form during the eclipse period. The other adsorbed particles were not uncoated and took no part in the process of infection. The maximal transfer of parental RNA to progeny virus was 4.5%.

Animals↗

Regulation of branched-chain aminoacyl-transfer ribonucleic acid synthetases in an ilvDAC deletion strain of Escherichia coli K-12.

Valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetase formation was compared in isogenic strains of Escherichia coli K-12 that differed only in that one strain carried a deletion of three genes of the ilv gene cluster, ilvD, -A, and -C. It was found that: (i) the activities of these synthetases in the deletion strain were less than those in the normal strain during growth in minimal medium supplemented with excess isoleucine, valine, and leucine, and (ii) their stability was reduced in the deletion strain during specific branched-chain amino acid limitations. The results of density-labeling experiments suggest that the in vivo stability of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetases requires some product missing in the ilvDAC deletion strain.

Amino Acyl-tRNA Synthetases↗

Ribonucleic acid and ribosomes of Bacillus stearothermophilus.

Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332-339. 1966.-The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10(-2)m MgCl(2)-10(-2)m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg(++) concentration to 10(-3)m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10(-2)m Mg(++) to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a T(m) at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a T(m) of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins.

Bacillus↗

INFECTIVITY OF HISTONE-POLIOVIRUS RIBONUCLEIC ACID PREPARATIONS.

Ludwig, Ernest H. (The Pennsylvania State University, University Park) and Christine E. Smull. Infectivity of histone-poliovirus ribonucleic acid preparations. J. Bacteriol. 85:1334-1338. 1963.-Some properties of histone-poliovirus ribonucleic acid (RNA) preparations, as relate to infectivity for HeLa cell monolayers, were investigated. The histone-RNA preparations were found to lose their infectivity rapidly at room temperature. They were considerably more stable at ice-bath temperature. Dilution of a histone-RNA preparation in a diluent containing an appropriate amount of histone yielded a plaque count which was proportional to the dilution factor, and which regressed linearly through the point of origin. Dilution of a histone-RNA preparation in a diluent containing no histone resulted in a rapidly decreasing plaque count which was not proportional to the dilution factor. The ability of histone to enhance the infectivity of poliovirus RNA was found to be dependent upon the presence of a low concentration of a monovalent salt in the histone-RNA preparations. Histone-RNA preparations containing approximately isotonic levels of NaCl exhibited a high degree of infectivity. When concentrations of NaCl outside this range were used, a great loss in infectivity of the histone-RNA preparations occurred. The NaCl could be replaced by KCl but not by CaCl(2), MgCl(2), or MgSO(4).

Animals↗

Length of the polyadenylated sequence in cytoplasmic ribonucleic acid isolated from fetal calf myoblasts differentiating in vitro.

Poly(adenylic acid) [poly(A)] containing cytoplasmic ribonucleic acid (RNA) in differentiating fetal calf myoblasts cultivated in vitro was examined by hybridization with radioactive poly(uridylic acid). The size distribution of the poly(A)-containing RNA after sucrose-gradient centrifugation was similar in cells before and after differentiation. There was no apparent correlation between the length of the poly(A) segment and the change in stability of messenger RNA which occurs on differentiation, nor with the polysomal or nonpolysomal localization of the RNA in the cytoplasm. The average length of the poly(A) segments in cytoplasmic RNA in the steady state was found to be dependent on the size of the RNA: the longer the RNA, the longer the average length of the poly(A) sequence. In contrast, in pulse-labeled RNa, the length of poly(A) is similar in all size classes of RNa.

Animals↗

Partial purification and characterization of a ribonucleic acid N2-guanine methyltransferase associated with avian myeloblastosis virus.

A nucleic acid methylase, N2-guanine ribonucleic acid (RNA) methyltransferase, which is associated with type C RNA tumor viruses, has been purified from avian myeloblastosis virions by gel filtration on Sephadex G-200, followed by chromatography on hydroxylapatite. The molecular weight estimated by gel filtration is 220 000, and the methylase activity has a pH optimum of 7.6--7.9. Magnesium and ammonium ions both stimulate activity 1.5-fold at 9.5 mM and 0.36 M, respectively, but apparently neither is essential for activity. Both daunomycin and adriamycin, antineoplastic drugs, also increase activity 1.5-fold at 1 mM. The enzyme was purified 120-fold from the virions and the activity is partially stabilized by dithiothretiol, but large losses were sustained during 24-h dialysis. The purified enzyme retains 75% of its activity on storage at -25 degrees C for 2 months in buffer containing 50% glycerol. Escherichia coli tRNAPhe and tRNAVal are preferred substrates with methylation occurring at position 10 of E. coli tRNAPhe.

Avian Leukosis Virus↗

Ribosomal ribonucleic acid (rRNA) gene typing for species identification.

Deoxyribonucleic acid (DNA) typing of ribosomal ribonucleic acid (rRNA) genes was performed with a polymerase chain reaction (PCR) assay for species identification. A variable region of the 28S ribosomal RNA gene was amplified with primers complementary to flanking sequences phylogenetically well conserved. The products of twelve animal DNAs (human, Japanese monkey, dog, cattle, pig, cat, rabbit, mouse, rat, chicken, frog, and fish) were separated by polyacrylamide gel electrophoresis, each revealing a few bands ranging from 150 to 100 base pairs. The band patterns obtained from each DNA sample differed in number and size, which indicates the applicability of the method to species identification. Samples containing either as little as 1 pg of DNA or degraded DNA of 0.2 to 0.5 kb in length were able to give detectable bands. Postmortem human tissue DNAs were tested as an example. They showed a pattern identical to the human control one, which was distinct from those of the other animals examined.

Animals↗

Ribonucleic acid synthesis dependent on exogenous triphosphates in nystatin-treated cells of of Kluyveromyces latis.

Kluyveromyces lactis cells treated with nystatin became permeable to ribonucleoside triphosphates. Although not viable, nystatin-treated cells were capable of sustaining ribonucleic acid synthesis. The system depended on the presence of divalent ions and the four nucleoside triphosphates, and was strongly stimulated by ammonium sulfate. The system utilized endogenous deoxyribonucleic acid as template. Synthesis of ribonucleic acid was associated with the cells and probably occurred internally.

Ammonium Sulfate↗

Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.

Escherichia coli↗

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences.

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.

Animals↗

Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: implications for secretion and function.

OBJECTIVE: Our purpose was to define the location and packaging of parathyroid hormone-related peptide in amnion-chorion and the potential target tissues for its action in fetal membranes. STUDY DESIGN: We studied fetal membranes by use of light microscopic immunocytochemistry with three monoclonal antibodies against distinct regions of the parathyroid hormone-related peptide molecule. For electron microscopy immunogold analysis with a monoclonal antibody specific to the 109-141 fragment was used to observe parathyroid hormone-related peptide intracellularly in amnion membrane and in the chorion layers. Multiplex reverse transcriptase-polymerase chain reaction with Southern blotting was used to identify parathyroid hormone/parathyroid hormone-related peptide receptor and control messenger ribonucleic acids in amnion and chorion-decidua. RESULTS: All monoclonal antibodies revealed immunoreactive parathyroid hormone-related peptide in the amniotic epithelial cells and in some fibroblast-like cells embedded in the extracellular matrix of the amnion. Parathyroid hormone-related peptide was also found in the chorion in fibroblast and trophoblast layers and in decidua. Ultrastructurally immunogold particles were evenly distributed throughout the amniotic epithelial cells and were present in apical microvilli and near the basal membranes. Electron microscopy studies of the chorion cytotrophoblast also showed freely dispersed immunogold particles of parathyroid hormone-related peptide with no packaging in secretory granules. Low to undetectable levels of parathyroid hormone/parathyroid hormone-related peptide receptor messenger ribonucleic acid were found in amnion tissue, whereas abundant receptor messenger ribonucleic acid was found in chorion-decidua. CONCLUSIONS: These results suggest the presence of a parathyroid hormone-related peptide paracrine system within the human fetal membranes.

Amnion↗

Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxin.

Group A streptococcal pyrogenic exotoxins (SPEs) A, B, and C and alpha-amanitin enhance host susceptibility to lethal endotoxin shock. The capacity of SPE C and alpha-amanitin to prepare rabbits for the enhancement phenomenon required pretreatment of the animals 1 to 2 h before giving endotoxin. Endotoxin clearance from the circulation of rabbits pretreated with either SPE C or alpha-amanitin was reduced. Even at the time of death, significant amounts of endotoxin remained in the circulation. It is proposed that the SPE and alpha-amanitin inhibit ribonucleic acid synthesis in Kupffer cells with concomitant alteration in reticuloendothelial clearnace function, allowing endotoxin to persist in the circulation and produce host injury. All three SPE types and alpha-amanitin inhibited ribonucleic acid synthesis by 50% or greater in whole liver cells. Kupffer cells, liver cell nuclei, and liver nuclear extracts; inhibition was observed liver cells from both mice and rabbits. The inhibitory effect by SPEs was dose dependent and was observed after as little as 15 min of preincubation with liver cells. The content of ribonucleic acid in liver nuclei of mice pretreated with either SPE C or alpha-amanitan was reduced, whereas total deoxyribonucleic acid and protein content remained unaltered.

Amanitins↗

Homology of ribosomal ribonucleic acid of Desulfovibrio species with Desulfovibrio vulgaris.

Three species of Desulfovibrio were found to have a high degree of ribosomal ribonucleic acid homology with Desulfovibrio vulgaris. Desulfotomaculum nigrificans, which is also a sulfate-reducing anaerobe, had only 38% ribosomal ribonucleic acid homology with D. vulgaris. The homologies of six other unrelated genera were determined and found to be lower than 50%.

Alcaligenes↗