Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Unspliced and partially spliced HIV RNAs are transported to the cytoplasm by the HIV encoded Rev protein. In the present study, a ribonucleoprotein complex which contains such incompletely spliced HIV RNA is identified. Soluble nuclear extracts were prepared from the lymphocyte cell line H9/IIIB that constitutively produces HIV-1 from a stably integrated provirus. Sucrose gradient centrifugation of the extracts and subsequent analysis of the gradient fractions by a ribonuclease protection assay revealed a population of incompletely spliced HIV-1 RNAs which accumulates in the 100S region of the gradient. Similar analysis of cellular mRNAs including beta-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) revealed that these RNA molecules also exhibit characteristic sedimentation profiles in sucrose gradients. This study suggests that nuclear ribonucleoprotein particles containing incompletely spliced HIV-1 RNAs are amenable for biochemical characterisation.
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PMID:Identification of a nuclear ribonucleoprotein particle which contains incompletely spliced HIV-1 RNAs. 1067 35

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native-state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native-state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far- and near-UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native-state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl-induced unfolding, increased in proportion to sucrose concentration. Native-state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange-resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large-scale than on small-scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native-state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.
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PMID:Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins. 1276 96

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%.
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PMID:INTRACELLULAR FATE OF MENGO VIRUS RIBONUCLEIC ACID. 1425 83

Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68- and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68- and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.
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PMID:Association of polyadenylation cleavage factor I with U1 snRNP. 1456 89

The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5' and 3' ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5' and 3' ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of approximately 50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.
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PMID:An in vitro model of hepatitis C virion production. 1570 97

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or deoxyhypusine synthase are essential for cell survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.
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PMID:Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex. 1621 87

Sucrose gradient profiles of polyribosomes from the coleoptilar node region of seedlings of Zea mays L. were obtained without pelleting and redispersion of the particles. Water stress caused a shift of ribosomes from the polymeric to the monomeric form, starting about 30 minutes after stress initiation and when the water potential of the tissue began to decrease measurably. After about 4 hours of stress (a decrease in tissue water potential of about 5 bars), most of the higher polymers of ribosomes had shifted to monoribosomes. Release of stress caused the ribosomes to revert from monomeric to polymeric form after a lag period apparently determined by the extent of prior stress. Use of bentonite and isolation of polyribosomes from combined stressed and control tissue gave results indicating that the reduced polyribosomal level was not an artifact caused by ribonuclease during isolation.Incubating roots in cycloheximide (2 micrograms per milliliter) had no effect on the proportion of polyribosomes in control roots, but it prevented the loss of polyribosomes caused by stress. Since cycloheximide inhibits the release of nascent polypeptide from polyribosomes, it appears possible that stress-effected loss in polyribosomes occurs only if polypeptides can be terminated and released.
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PMID:Rapid Changes in Levels of Polyribosomes in Zea mays in Response to Water Stress. 1665 50

Changes in the content of starch, protein, and RNA and in the activity of their hydrolases in the rice endosperm (Oryza sativa L., variety IR8) were determined during the first week of germination without added nutrient both in the dark and in the light. Changes were generally more rapid in the dark than in the light. Oxygen uptake and RNase activity started to increase and the root protruded on the second day, followed by the coleoptile on the third day, and the primary leaf on the fourth day. ATP level was at a maximum on the fourth day. The activity of amylases and R enzyme increased progressively, but that of phosphorylase tended to decrease during starch degradation. A new alpha amylase isozyme band appeared during germination. Glucose was the major product of starch degradation. Sucrose, maltose, maltotriose, raffinose, and fructose were also detected. Protease activity reached a maximum on the fifth or sixth day and closely paralleled the increase in soluble amino N and soluble protein.In embryoless seed halves with 0.12 muM gibberellin As, peak protease activity occurred in 2.5 days and peak alpha amylase activity on the fifth day of incubation. The production of alpha amylase, protease, and R enzyme was inhibited by 40 muM cycloheximide, but only alpha amylase and R enzyme were inhibited by 20 mug/ml actinomycin D.
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PMID:Biochemical Changes in the Rice Grain during Germination. 1665 42

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.
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PMID:Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY. 1666 Nov 78

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.
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PMID:Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION. 1666 93


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