Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribozymes that target specific messenger RNA transcripts are powerful tools in the emerging fields of functional genomics, proteomics, and metabolomics. We have found that successful in vitro testing greatly increases the likelihood of producing ribozymes with good efficacy in living cells. A rapid and simple nonradioactive method for systematic in vitro testing of ribozyme-cleaving activity is reported. Ribozymes are synthesized enzymatically from double-stranded DNA (dsDNA) oligonucleotides without vector cloning. Substrate target DNA template is cloned into a vector flanked with SP6 and T7 promoters at multiple cloning sites that permit colorimetric screening and ampicillin selection, enhancing the efficiency of the cloning procedure. Ribozyme cleavage products are satisfactorily resolved on 2.0% NuSieve 3:1 agarose (FMC Products, Rockland, ME)/formaldehyde gels by electrophoresis. This method avoids the preparation of polyacrylamide gels. Using this procedure, the ribozyme, target substrate RNA, and ribozyme cleavage products are all easily detected by ethidium bromide staining. Resolution and detection are fast and simple, eliminating the need for either polyacrylamide gel analysis or radiolabeling. The use of RNase inhibitors in the assays is also assessed and discussed.
 ...
PMID:A rapid and simple nonradioactive method for in vitro testing of ribozyme activity. 1223 17

The genomic DNA of bacteria is highly localized in one or a few bodies known as nucleoids. A number of restraints to the unfolding of the DNA of spermidine nucleoids from Escherichia coli were previously associated with characteristic urea concentrations (U(m) values). The dominant restraint to unfolding was sensitive to pancreatic RNase and underwent a cooperative transition at U(m) = 3.2 M urea. The losses of the RNase-sensitive restraint caused by urea or pancreatic RNase appear to result from breakage of cotranslational insertion linkages which joined the nucleoid to the cell envelope in growing cells. This conclusion is based upon effects from exposures of cells to antibiotics (chloramphenicol, rifampicin, streptomycin), treatment of nucleoid preparations with formaldehyde or concentrated NaCl solutions, and effects of urea on purified ribosomes. The specific RNase-sensitive and urea-sensitive components of the spermidine nucleoids are suggested to be the mRNA and ribosomes, respectively, of cotranslational insertion linkages.
 ...
PMID:Hypothesis: the RNase-sensitive restraint to unfolding of spermidine nucleoids from Escherichia coli is composed of cotranslational insertion linkages. 1248 11

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.
 ...
PMID:INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS. II. ENZYMATIC AND OTHER HYDROLYTIC TREATMENTS. 1408 39

We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.
 ...
PMID:Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy. 1793 84

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.
 ...
PMID:Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking. 1807 27

Potato leafroll virus (PLRV) was purified from infected potato (Solanum tuberosum L.) with yields of 0.4-0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 x 10(6) and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.
 ...
PMID:Purification and characterization of potato leafroll virus. 1863 9

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H(2) and formaldehyde-D(2) and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d(0)- and d(4)-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.
 ...
PMID:A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry. 2256 Feb 80

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.
...
PMID:Staufen1 senses overall transcript secondary structure to regulate translation. 2433 23

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and posttranslational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MNase) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including PCR, qPCR, or sequencing. This protocol outlines each of these steps for both yeast and human cells.
 ...
PMID:Chromatin Immunoprecipitation in Human and Yeast Cells. 2952 40


<< Previous 1 2 3 4