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)

Activity of various natural and synthetic polynucleotides as a carrier for streptolysin S was tested in a resting cell system. As the carrier, intact molecules of MS2 RNA, E coli tRNA or rat liver RNA were almost inactive, whereas RNase I core of these RNAs, especially the core fractions eluted from DEAE-cellulose column at higher NaCl concentrations, effectively induced production of the extracellular hemolysin. The carrier activity of yeast RNA was significantly enhanced by simultaneous addition of RNase I, to the streptococcal suspension. Preincubation of yeast RNA with growing streptococci or a protein fraction from the bacterial culture supernatant increased its carrier activity. Evidences were obtained suggesting involvement of streptococcal nuclease in the enhancement of the carrier effect. Production of the streptococcal hemolysin was markedly promoted by polyguanylic acid but neither by polyadenylic acid, polycytidylic acid nor by polyuridylic acid. Like trypan blue, polyguanylic acid exerted potent inhibitory effect on hemolytic activity of streptolysin S complex. Similar but less marked effect was observed with certain RNase I core fractions of rat liver RNA. Some data concerning effect of polynucleotides on stability of the hemolysin were also presented.
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PMID:Effects of polynucleotides on production and activity of streptolysin S. 615 93

The limited hydrolisis of bacteriophage MS2 RNA by nuclease S1 and ds-specific snake venom RNase was studied in a wide range of ionic strength, at different pH, after heating and (slow and fast) cooling and at various enzyme-substrate relations. It was shown that the RNA has exposed hydrolisis sites for both nucleases. The localizations of these sites are very specific and are not altered in all conditions studied. The hydrolisis rate was changed in some conditions, at that the fragments patterns in denaturing electrophoresis did not move. It was supposed that the RNA has strongly predetermined and predominant conformation which could not be altered by strong influences.
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PMID:[Study of spatial organization of the RNA of phage MSZ using nucleases specific for the secondary structure]. 629 1

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.
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PMID:Formation of bacteriophage MS2 infectious units in a cell-free translation system. 895 35

A full-length cDNA copy of the RNA genome of bacteriophage MS2 was assembled by the in-frame ligation of the central portion of the genome into a plasmid containing the 5' and 3' ends. Upon transformation of the ligation reaction into Escherichia coli, infectious phage particles were released into the medium. The plaquing ability of the phage produced from the cDNA construct was assessed against various bacterial strains confirming that the bacteriophage produced were male-specific. Sensitivity to RNase in agar overlay was used to confirm that the phage contained RNA. In addition, the phage were unable to infect piliated cells overexpressing MS2 coat protein, a resistance conferred by the binding of recombinant coat protein to the infecting strand of RNA at the replicase initiation region, thus preventing translation of the replicase gene. The phage capsids were visualised after negative staining by transmission electron microscopy, and appeared as spherical particles of approximately 25 nm diameter. The capsid proteins were examined by Western blotting, confirming the presence of a single protein of approximately 14 kDa, which bound anti-MS2 coat protein antibodies. The genomic RNA from single plaques was analysed by reverse transcription-PCR and the presence of the MS2 coat protein gene confirmed by DNA sequencing. The production of replicative MS2 phage from cDNA fragments was used to assess the viability of MS2 coat protein mutants, which had previously been shown to assemble into T = 3 capsid-like particles when expressed in vivo from a bacterial vector. The E76D mutation did not appear to affect phage viability, whilst replacement of the completely conserved P78 residue with asparagine abolished the production of infectious particles, suggesting that P78 may be involved in interactions with the phage maturation protein.
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PMID:Analysis of phage MS2 coat protein mutants expressed from a reconstituted phagemid reveals that proline 78 is essential for viral infectivity. 905 64

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper. (a) Collisional activation of permethylated oligosaccharide molecular ions (MS2) as illustrated by maltoheptaose, produces abundant fragments from glycosidic bond cleavages which indicate composition and sequence, and weak cross-ring cleavage products which denote specific linkages within the oligosaccharide. Through the trapping and further dissociation of these fragments (MSn), cross-ring cleavage products can be confirmed and their relative abundances increased to facilitate interpretation. (b) The mechanisms of formation of two isobaric ions or ions isobaric with another ion's isotope peaks, such as those present in the MS2 spectrum of the ribonuclease B oligosaccharide GlcNAc2-Man5 can be independently established by separate MS3 experiments. (c) Ions in the MS2 spectrum, specific for individual components of an isobaric mixture, can be isolated and characterized by further stages of fragmentation. This is illustrated by two isobaric oligosaccharides from chicken ovalbumin of the composition HexNAc5Hex5. These findings indicate the utility of ion trap mass spectrometry towards the facile determination of oligosaccharide composition, sequence, branching and linkage, providing a wealth of structural information not obtainable by other individual methods of carbohydrate mass spectrometric analysis.
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PMID:Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry. 933 19

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.
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PMID:Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards. 981 78

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.
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PMID:TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening. 1172 36

A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.
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PMID:Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H. 1236 83

Glycosylation is one of the most important posttranslational modifications affecting the functions of proteins and cell activities. Mass spectrometry (MS) has proven to be an effective tool for structural glycobiology and has helped gain an understanding of glycoprotein-mediated diseases. Although electro-spray ionization-tandem MS remains widely recognized as an effective means for oligosaccharide characterization, the hydrophilic nature of glycans has often caused the poor ionization efficiency requiring either derivatization or nanoelectrospray to improve detection sensitivity. In this report we describe the use of a chip-based infusion nanoelectrospray platform coupled with the hybrid triple quadrupole/linear ion trap for identification and characterization of glycosylation in complex mixtures. The high-mannose-type N-glycosylation in ribonuclease B was used to map the glycosylation site and obtain glycan structures. Using the chip-based nanoelectro-spray with precursor ion scanning linear ion trap MS, we were able to map the glycosylation site and obtain the glycan structures in ribonuclease B at 100 fmol/microL in a single analysis. In addition, a new, low-abundant glycoform with an additional hexose (Hex10GlcNAc2) attached to ribonuclease B was discovered. The results reported here demonstrate that the chip-based infusion nanoelectrospray ionization coupled to a quadrupole/linear ion trap platform is a valuable system, as it provides high sensitivity and stability for nanoelectrospray analysis, and allows extended acquisition time for completing precursor ion scanning and subsequent MS2 and MS3 information in a single analysis.
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PMID:Characterization of protein glycosylation using chip-based nanoelectrospray with precursor ion scanning quadrupole linear ion trap mass spectrometry. 1646 44

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.
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PMID:RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system. 1830 35


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