Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The physicochemical properties of Venezuelan equine encephalomyelitis (VEE) virus and of its ribonucleoprotein (RNP) were studied. Upon purification in a discontinuous or linear sucrose gradient, the losses of infectivity were small (25%), and 96% of cellular proteins were removed. The purified virus was homogeneous with respect of sedimentation rate (s20, w = 265 S). The CsCl gradient was unsuitable for purification of infectious virus because the latter was destroyed at high CsCl concentrations. Buoyant density of the virus in CsCl after formaldehyde fixation was 1.21--1.22 g/cm3. Treatment of the virus with 1% Nonidet P-40 proved to be the most effective method for isolation of the RNP. The structures thus obtained contained practically all of the viral RNA and about 20% of viral proteins, and were homogeneous with respect of sedimentation rate (153 S) and buoyant density (1.40--1.42 g/cm3 in CsCl after formaldehyde fixation). The RNPs were sensitive to ribonuclease.
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PMID:Properties of virions and ribonucleoproteins of Venezuelan equine encephalomyelitis virus. 1 19

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.
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PMID:Proton-magnetic-resonance studies of the lysine residues of ribonuclease A. 23 43

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.
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PMID:A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum). 52 78

Even a small amount of formaldehyde is shown to induce a drop in the RNase A enzymatic activity. This drop is rapid from the start and then begins to be slower. A supposition was made on nature of the enzyme activity. Comparison of the effects of formaldehyde on the enzymatic and the destabilizing activity of RNase A was made. The effect of formaldehyde on the enzymatic activity does not correlate with its effect on the ability of RNase to destabilize the DNA double helix.
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PMID:[Effect of formaldehyde on the enzymatic activity of RNAase A]. 56 74

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.
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PMID:The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells. 98 39

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.
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PMID:Physical and chemical characterization of purified ovalbumin messenger RNA. 115 96

The single-strand specific nuclease S1 from Aspergillus oryzae (EC 3.1.4.21) was purified 600-fold in 16% yield from dried mycelia. Determination of the isoelectric point of S1 nuclease as 4.3-4.4 allowed adjustment of chromatographic conditions such that the enzyme was isolated free of contaminating ribonucleases T1 and T2. S1 nuclease so purified was used for removal of single-stranded portions from the RNA of the Escherichia coli phage MS2, which has a helical content of about 65% in vitro. At 23 degrees, increasing amounts of enzyme converted the RNA to mononucleotides in about equimolar base ratios. No small intermediates of chain length 2-8 were found. At 0 degrees, MS2 RNA hydrolysis was slower and reached, in exhaustive digests, a plateau where 70% of the substrate RNA remained insoluble in 66% EtOH. With [32P]MS2 RNA, strip chart counting of 6% acrylamide-6 M urea electrophoresis patterns of such digests gave recoveries of 80-91% in the form of defined oligomer bands. On 2.5% acrylamide-0.5% agarose gels, the molecular weights of the major oligomers were found to range from 25,000 to 41,000. Similar to purified tRNAArg used as a control, these oligomers were not resistant to pancreatic RNase-RNase T1 hydrolysis at 37 degrees, and were not bound on hydroxylapatite at 50 degrees in 0.14 M sodium phosphate (pH 6.8). Melting of the oligomers gave complex profiles without a clear Tm and showed an increase in A260 of 35% at 93 degrees over that at 28 degrees. Upon formaldehyde denaturation of MS2 RNA prior to S1 nuclease hydrolysis, no resistant oligomers were found.
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PMID:S1 nuclease hydrolysis of single-stranded nucleic acids with partial double-stranded configuration. 118 98

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.
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PMID:Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells. 165 2

The 43-kDa non-O antigenic component isolated from the crude ribosomal fraction of Salmonella typhimurium [9] was further purified by affinity chromatography (43-kDa protein: 43-kDp). Immunization with 43-kDp did not induce complete mouse protection in CF1 mice to 500 LD50 of S. typhimurium, although it elicited a substantial IgG antibody response. The 43-kDp exhibited the mitogenicity to splenocytes (CF1 and C3H/HeJ) and B cell-rich populations (CF1). Complexing 43-kDp with the compact ribosomes of Streptococcus pyogenes by formaldehyde (complex vaccine: CV) elicited both IgM and IgG antibodies to 43-kDp. CV induced a boosting effect to enhance IgG antibody response. Moreover, CV generated delayed-type hypersensitivity to salmonella antigens and also conferred complete protection against 500 LD50 challenge of S. typhimurium to CF1 mice. These abilities of CV were reduced or impaired by RNase digestion. CV was able to induce partial or complete protection in inbred mouse strains (C3H/HeN, C3H/HeJ, DBA/2 and A/J). These data, in addition to other reports, suggest that conformational stability between ribosomes and contaminating substances such as 43-kDp or O-antigens might be required for the overall effects of the ribosomal vaccine.
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PMID:Requirement of the conformational stability of a Salmonella ribosomal vaccine for its mouse protection. 193 Nov 35

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.
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PMID:Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate. 244 24


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