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)

RNase protection assays (RPA) employing multiprobe sets are powerful tools to simultaneously measure transcription of several different genes. We used BD Biosciences/Pharmingen's mouse chemokine probeset mCK-5c to measure chemokine gene expression in brain and spleen tissue of mice. Depending on the RPA protocol used, we observed differences in the relative amounts of transcripts for interferon-inducible protein 10 (IP-10) and T-cell activation-3 (TCA-3). Isolation and sequencing of the IP-10 specific gene from the mCK-5c probeset revealed two nucleotide insertions in the probe that are not present in the natural IP-10 cDNA. We show that these insertions cause RNase A-dependent degradation of the protected IP-10 mRNA yielding a fragment indistinguishable in size from that specific for TCA-3, thus leading to over-interpretation of TCA-3 expression as well as underestimation of IP-10 gene expression levels.
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PMID:The IP10 (CXCL10) specific cDNA probe of the mCK-5c multiprobe RNase protection assay kit carries two nucleotide insertions that complicate the interpretation of results. 1822 43

This appendix describes the preparation of selected bacterial media and of buffers and reagents used in the manipulation of nucleic acids and proteins. Recipes for cell culture media and reagents are located elsewhere in the manual. RECIPES: Acids, concentrated stock solutions; Ammonium acetate, 10 M; Ammonium hydroxide, concentrated stock solution; ATP, 100 mM; BCIP, 5% (w/v); BSA (bovine serum albumin), 10% (100 mg/ml); Denhardt solution, 100x; dNTPs: dATP, dTTP, dCTP, and dGTP; DTT, 1 M; EDTA, 0.5 M (pH 8.0); Ethidium bromide solution; Formamide loading buffer, 2x; Gel loading buffer, 6x; HBSS (Hanks balanced salt solution); HCl, 1 M; HEPES-buffered saline, 2x; KCl, 1 M; LB medium; LB plates; Loading buffer; 2-ME, (2-mercaptoethanol)50 mM; MgCl(2), 1 M; MgSO(4), 1 M; NaCl, 5 M; NaOH, 10 M; NBT (nitroblue tetrazolium chloride), 5% (w/v); PCR amplification buffer, 10x; Phosphate-buffered saline (PBS), pH approximately 7.3; Potassium acetate buffer, 0.1 M; Potassium phosphate buffer, 0.1 M; RNase a stock solution (DNase-free), 2 mg/ml; SDS, 20%; SOC medium; Sodium acetate, 3 M; Sodium acetate buffer, 0.1 M; Sodium phosphate buffer, 0.1 M; SSC (sodium chloride/sodium citrate), 20x; SSPE (sodium chloride/sodium phosphate/EDTA), 20x; T4 DNA ligase buffer, 10x; TAE buffer, 50x; TBE buffer, 10x; TBS (Tris-buffered saline); TCA (trichloroacetic acid), 100% (w/v); TE buffer; Terrific broth (TB); TrisCl, 1 M; TY medium, 2x; Urea loading buffer, 2x.
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PMID:Common buffers, media, and stock solutions. 1842 17

Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17-18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29-1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200-300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).
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PMID:Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells. 1862 47

Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17 degrees C. From the 7,400 mutants tested, 11 displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA), but it also appears to possess novel modes of cold adaptation.
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PMID:Mediators of lipid A modification, RNA degradation, and central intermediary metabolism facilitate the growth of Legionella pneumophila at low temperatures. 1976 2

The sequence of poly(adenylic) acid, present at the 3' end of the majority of eukaryotic mRNA molecules, forms the basis of a sensitive technique for the estimation of mRNA content in nucleic acid samples. Under suitable conditions, poly(A) will form RNA-RNA hybrids with poly(U) in vitro. The poly (A) content of RNA samples can therefore be detected by hybridization with saturating amounts of (3)H-poly(U) (1,2). Following the removal of excess (3)H-poly(U) by ribonuclease treatment, the hybrids can be collected by TCA precipitation and quantified by scintillation counting. If the results are compared with data obtained from a parallel experiment using known amounts of poly (A), a value for the poly (A) content of any number of RNA preparations can be obtained. The technique can be used to detect less than 10(-10)g of poly(A).
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PMID:The estimation of mRNA content by poly(u) hybridization. 2137 82

The data presented here are related to the research paper entitled "Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses" (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.
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PMID:TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data. 2987 49


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