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Query: EC:3.1.27.4 (ribonuclease)
 6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conventional methods (i.e. gradient centrifugation) for the purification of oncornaviruses are usually not effective in complete removal of nonviral proteins. Such contaminants often prove to be a nuisance in subsequent immunological or biochemical studies. Hyperimmune sera prepared from these viruses must be absorbed to assure specificity; cell-derived proteins can be shown to interfere with studies of virus structural proteins, nucleic acids, or viral enzymes. Herein is described a method for removal of most of these contaminants. Viruses are diluted in a high concentration of NaCl to achieve a final concentration of 15%, incubated for 30 min, sedimented, and resuspended in buffer. This procedure results in reductions of up to 48% of the protein without affecting particle count. Immunological, biochemical, and biological properties are not adversely affected. Of the proteins removed, fetal calf serum components and a ribonuclease (presumably cell-derived) were identified. This technique differs significantly from other high-salt methods in that the virus is not precipitated from suspension. It is believed that absorbed proteins are desorbed and left in solution (or suspension) as the virus is sedimented by centrifugation.
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PMID:New method for the removal of extraneous proteins from purified oncornaviruses. 5 58

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three catagories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion. Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (deltaH) accompanied by either an increase or no change in the temperature of transition (Tc). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region. Cytochrome c and A1 protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both Tc and deltaH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer. Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the Tc but did induce a linear decrease in the magnitude of the deltaH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a deltaH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.
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PMID:Effects of proteins on thermotropic phase transitions of phospholipid membranes. 5 74

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

Fidelity of preribosomal RNA transcription in vitro was studied after selective deproteinization of nucleoli using either sequential salt extraction or sodium deoxycholate treatment. Homochromatography fingerprinting and identification of marker oligonucleotides from a T1 ribonuclease digest of the transcripts were used to evaluate the RNA products. These studies indicated that: (1) nucleoli retained their endogenous RNA polymerase I activity and the specificity of transcription up to 0.6 M NaCl extraction; (2) exogenous RNA polymerase I transcribed nucleolar chromatin only after 1.0 M NaCl extraction and the transcription pattern, like that of totally deproteinized DNA, was completely random; (3) extraction of nucleoli with deoxycholate resulted in a DNP complex in which the endogenous RNA polymerase I transcribed pre-rRNA specifically; however, it also initiated random transcription, producing a "mixed" fingerprint pattern on the homochromatogram. The random transcription was selectively inhibited either by deoxycholate or rifampicin AF/013. These studies indicate that the selectivity of pre-rRNA transcription is due both to the endogenous RNA polymerase I molecules that were involved in transcription in vivo and are tightly bound to the template and to factors in intact nucleoli which prevent random transcription by the released RNA polymerase I molecules.
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PMID:Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization. 11 95

When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
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PMID:Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. 16 2

Treatment of neuroblastoma cells with dibutyryl-adenosine 3':5'-monophosphate or adenine induced axon formation and a three-fold increase in the polyadenylate, poly(A), content of the polysomal mRNA. The extracted poly(A) contained 90% adenylic acid and showed a mobility of 6--7 S in dodecylsulfate-polyacrylamide gel electrophoresis. Treatment with dibutyryl-adenosine 3':5'-monophosphate or adenine, also induced a 4--6 fold increase in a nuclear enzymic activity that incorporated [3H]ATP to an acid-insoluble polymer in a cell-free system. This polymer, like poly(A) extracted from the polysomal mRNA, was bound at high salt concentration to nitrocellulose filters. [3H]ATP incorporation was Mg2+-dependent, sensitive to ribonuclease and EDTA and resistant to deoxyribonuclease and actinomycin D. There was no incorporation of [3H]UTP or [3H]dTTP and addition of TUP, CTP and GTP did not increase the incorporation of [3H]ATP. 5-Bromodeoxyuridine induced axon formation of neuroblastoma cells and poly(A) polymerase activity, without increasing the poly(A) content in the polysomal mRNA. The results indicate that induction of axon formation of neuroblastoma cells is associated with an increase in the activity of poly(A) polymerase. It is suggested that the induction of this enzyme may be generally involved in cell differentiation.
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PMID:Induction of polyadenylate polymerase and differentiation in neuroblastoma cells. 17 99

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.
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PMID:Demonstration of an androgen receptor in rat pancreas. 18 42

Replicative intermediate (RI), replicative form (RF) and single-stranded (SS) RNA have been isolated from BHK cells infected with a bovine enterovirus by salt precipitation and gel filtration techniques. Kinetic experiments showed that at no time up to 16 h post-infection (p.i.) did the amount of RF exceed that of RI or SS RNA. Electrophoresis of RF on 1.5% polyacrylamide-agarose gels showed that at least three species of double-stranded RNA were present, one of which was associated with an accessible poly(A)-containing tract. All of the RF was denatured by 99% dimethylsulphoxide (DMSO), although reannealling occurred rapidly when samples were returned to aqueous conditions. No evidence for circular structures in the RF molecular population was found by use of caesium sulphate density gradients containing ethidium bromide. Treatment of RI with ribonuclease produced double-stranded RNA molecules, some of which were smaller in size than intact RF. Denaturation with DMSO and analysis on 99% DMSO sucrose gradients showed that the RI did not contain single strands of greater length than virion RNA. A portion of the RI bound to poly(U)-Sepharose 4B columns. The poly(A) tracts involved were present only in the nascent RNA strands with greatest sedimentation coefficients (30 to 35S). Bovine enterovirus induced SS RNA was heterogeneous with regard to both sedimentation through sucrose gradients and mobility on acrylamide gels compared to purified virion RNA. The reason for this difference has never been satisfactorily resolved. Sedimentation through 99% DMSO-sucrose gradients showed that the heterogeneity was due to aggregation rather than any variation in chain length or conformational differences. Our results support the single-stranded template model rather than a circular model for picornavirus RNA replication.
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PMID:Studies of the replication of a bovine enterovirus RNA. 22 21

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.
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PMID:Release of template restriction in chromatin by nuclear 4.5s RNA. 32 18

The aim of our work was to evaluate the diagnostic value of the determination of the ribonuclease activity in sera of patients with gynaecologic malignomas. We therefore developed an assay for ribonuclease activity. In the course of optimization of the assay conditions we investigated the applicability of 4 commercially available RNA-preparations as substrate and the dependency of the ribonuclease activity on salt-concentration. The ribonuclease activity of 42 representative female patients (12 controls, 11 with ovarian carcinoma, 10 with corpus carcinoma, 9 with collum carcinoma) is presented.
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PMID:[Serum ribonuclease activity in patients with gynaecologic malignomas (author's transl)]. 51 Aug 96


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