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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated with the heavy chains.
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PMID:Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. 923 2

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.
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PMID:The isolated RNase H domain of murine leukemia virus reverse transcriptase. Retention of activity with concomitant loss of specificity. 926 41

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
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PMID:Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1. 937 37

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by progressive ataxia, telangiectasia, sinopulmonary infections, hypersensitivity to ionizing radiation, and combined immunodeficiency. Recently, the AT gene (ATM) was cloned and shown to be mutated in AT patients. In this report, mutation analysis of ATM was performed in a 24-year-old AT patient without immunodeficiency. ATM amplified with reverse transcriptase-polymerase chain reaction (RT-PCR) was screened with a ribonuclease (RNase) cleavage assay and auto-sequenced. This patient, a compound heterozygote, showed two mutations in ATM: one missense mutation leading to a Leu2656Pro substitution and the other to the truncation at codon 3047 (Arg-->ter). The latter mutation is within the phosphatidylinositol 3-kinase (PI 3-kinase)-like domain and the former is outside but close to the domain. The particular phenotype in our patient, no immunodeficiency, suggests incomplete functional loss of ATM protein. The clinical spectrum of AT caused by ATM mutations may be broader than previously thought. Further analysis of patients with similar phenotypes will make the relation between ATM genotype and phenotype clear.
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PMID:Ataxia-telangiectasia without immunodeficiency: novel point mutations within and adjacent to the phosphatidylinositol 3-kinase-like domain. 945 Aug 74

New data are presented on the interaction of model synthetic peptides containing an arginine-rich region of human immunodeficiency virus (HIV-Tat), with native RNA molecules: tRNA(Phe) of Saccharomyces cerevisiae and 5S rRNA from Lupinus luteus. Both RNA species form complexes with the Tat1 (GRKKRRQRRRA) and Tat2 (GRKKRRQRRRAPQDSQTHQASLSKQPA) peptides, as shown by electrophoretic gel shift and RNase footprint assays, and CD measurements. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe as well as in the loop D of 5S rRNA is specifically protected against RNases. Our data indicate direct interactions of guanine of RNA moieties with arginine residues. These interactions seem similar to those observed in DNA-protein complexes, but different from those previously observed in the TAR RNA-Tat complexes.
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PMID:Interaction of HIV Tat model peptides with tRNA and 5S rRNA. 951 68

The existence of retroviral reverse transcriptases as monomers or dimers is rather intriguing. A classical example of the former is murine leukemia virus reverse transcriptase (MuLV RT), while human immunodeficiency virus type 1 (HIV-1) RT represents the latter. A careful scrutiny of the amino acid sequence alignment of the two enzymes pinpoints the region tentatively responsible for this phenomenon. We report here the construction of a chimeric enzyme containing the first 425 amino acid residues from the N-terminal domain of HIV-1 RT and 200 amino acid residues from the C-terminal domain of MuLV RT. The chimeric enzyme exists as a monomer with intact DNA polymerase and RNase-H functions.
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PMID:An enzymatically active chimeric HIV-1 reverse transcriptase (RT) with the RNase-H domain of murine leukemia virus RT exists as a monomer. 954 16

The 2-5A system is an RNA degradation pathway that can be induced by the interferons (IFNs). Treatment of cells with IFN activates genes encoding several double-stranded RNA (dsRNA)-dependent synthetases. These enzymes generate 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A) from ATP. The effects of 2-5A in cells are transient since 2-5A is unstable in cells due to the activities of phosphodiesterase and phosphatase. 2-5A activates the endoribonuclease 2-5A-dependent RNase L, causing degradation of single-stranded RNA with moderate specificity. The human 2-5A-dependent RNase is an 83.5 kDa polypeptide that has little, if any, RNase activity, unless 2-5A is present. 2-5A binding to RNase L switches the enzyme from its off-state to its on-state. At least three 2',5'-linked oligoadenylates and a single 5'-phosphoryl group are required for maximal activation of the RNase. Even though the constitutive presence of 2-5A-dependent RNase is observed in nearly all mammalian cell types, cellular amounts of 2-5A-dependent mRNA and activity can increase after IFN treatment. One well-established role of the 2-5A system is as a host defense against some types of viruses. Since virus infection of cells results in the production and secretion of IFNs, and since dsRNA is both a frequent product of virus infection and an activator of 2-5A synthesis, the replication of encephalomyocarditis virus, which produces dsRNA during its life cycle, is greatly suppressed in IFN-treated cells as a direct result of RNA decay by the activated 2-5A-dependent RNase. This review covers the organic chemistry, enzymology, and molecular biology of 2-5A and its associated enzymes. Additional possible biological roles of the 2-5A system, such as in cell growth and differentiation, human immunodeficiency virus replication, heat shock, atherosclerotic plaque, pathogenesis of Type I diabetes, and apoptosis, are presented.
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PMID:The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation. 962 81

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.
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PMID:Degradation of hammerhead ribozymes by human ribonucleases. 964 39

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
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PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.
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PMID:Mutations of the CD40 ligand gene and its effect on CD40 ligand expression in patients with X-linked hyper IgM syndrome. 1048 40


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