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)

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.
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PMID:Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase. 169 Oct 20

HIV (human immunodeficiency virus) viraemia in serum or plasma of HIV-infected individuals was investigated by the polymerase chain reaction assay (PCR) in combination with reverse transcription to detect HIV-1 genomic RNA. Before PCR, plasma or serum was ultracentrifuged, precipitated virions were then treated with a RNase-free DNase, and a cDNA from the HIV-1 genomic RNA was synthesized. Thirty-three fresh plasma and seven sera from either HIV-1 antibody-positive individuals or patients treated with AZT were tested. Plasma from three patients were assayed 3 or 6 months apart. Twelve sera from HIV-1 antibody-negative individuals were used as negative control. PCR was performed with primers in LTR, gag and env regions: 11 of 40 samples were positive with three primer pairs, 16 with two primer pairs and 11 with only one primer pair. PCR on HIV-1 genomic cDNA was positive in 38 out of the 40 plasma or serum samples (95%), regardless of the clinical stage of the infection: HIV-1 was detected in 14 of the 15 untreated subjects and in 24 of the 25 AZT-treated patients. HIV p24 antigen was detected in the serum of 38% of subjects (15 of 40). The results suggest that this method is suitable for the detection of viral particles in plasma or serum from HIV-1-infected individuals irrespective of antiviral treatment.
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PMID:The polymerase chain reaction for the detection of HIV-1 genomic RNA in plasma from infected individuals. 171 16

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase.
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PMID:A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. 171 68

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.
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PMID:Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions. 182 47

The trans-acting response element (TAR) within the long terminal repeat of human immunodeficiency virus (HIV) is present in all 5' termini of HIV mRNAs and is recognized by the viral Tat protein. Now we describe that the 59-nucleotide-long TAR-RNA exists as a ribonucleoprotein particle in polysomal and heterogeneous nuclear RNP fractions of HIV-1-infected HeLa-T4+ cells. Applying an immunoprecipitation technique this Tat.TAR complex could be isolated from total cell extracts as well as from polysomal or heterogeneous nuclear RNP fractions. The chain length and the identity of the TAR-RNA were established by RNase protection assays while the Tat protein was confirmed by Western blotting technique. The TAR-RNA in this complex was sequenced and found to comprise nucleotides +2 to +61 and hence includes the 3-nucleotide bulge (nucleotides +23 to +25) and the loop sequence of the TAR stem-and-loop structure. The Tat.TAR complex is present in cells at low abundance (12.5 x 10(3) copies/cell). In contrast to the TAR-containing mRNAs, which decay very rapidly after incubation of cells with actinomycin D (half-life of approximately 120 min) the half-life of TAR in the Tat.TAR complex is greater than 180 min. Alignment studies revealed that TAR-RNA (positive strand) has a potential binding ability to the U5 region within the long terminal repeat (DNA negative strand; nucleotides +107 to +147); a complementary binding with a continuous homology of 16 nucleotides was identified. It is proposed that the Tat.TAR complex functions as a small ribonucleoprotein particle during transcription initiation of HIV mRNA.
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PMID:Formation of a small ribonucleoprotein particle between Tat protein and trans-acting response element in human immunodeficiency virus-infected cells. 183 May 89

We have studied human immunodeficiency virus type 1 (HIV-1) infection in three different human neuroblastoma cell lines; SK-N-MC, IMR-32 and SH-SY5Y. In all of these cell lines the infection became productive. However, the virus expression was different as determined by the p24 antigen capture assays from culture supernatants and immunochemical (APAAP) staining of cells. The medium of SK-N-MC cells contained approximately 300 pg p24 antigen per 10(6) cells, 0.1-1% of the cells were p24 antigen-positive and characteristic genomic and subgenomic HIV mRNA species were seen in Northern blotting. In infected IMR-32 and SH-SY5Y cell cultures, the HIV-1 production was below the level of detection. However, infectious virus was found by inoculating cultures of the lymphoid cell C8166 with the cell-free supernatant fluid from the neuroblastoma cultures. The lymphoid cells became positive within one week. Moreover, phytohemagglutinin-stimulated normal human lymphocytes produced virus, if cocultured with any of the three infected neuroblastoma cell lines. The infection was persistent and has been followed, using the above techniques, for 4 months in the case of SK-N-MC and IMR-32 cells and 6 months in the case of SH-SY5Y cells. During this period, no alterations in cell morphology, viability, or proliferative capacity were seen. All three neuroblastoma lines were negative for the CD4 receptor mRNA according to Northern hybridization and RNase protection assays. We conclude that HIV-1 produces persistent and inapparent infection in human neuroblastoma cells, using a CD 4-independent mechanism of entry to the cells.
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PMID:Persistent inapparent HIV-1 infection of human neuroblastoma cells. 195 29

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.
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PMID:Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry. 197 May 76

The specific interaction between a defined structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.
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PMID:Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element. 199 59

The RNase A mismatch cleavage method has been applied to the characterization of natural genetic variation of human immunodeficiency virus (HIV) from different geographical areas. The approach provides a rapid and simple assay for the analysis of differences in closely related viral isolates and allows the establishment of phylogenetic relationships between epidemiologically distinct viruses. Our results show a broad clustering of circulating viruses according to their geographical distribution. We also have analyzed the temporal appearance of mutations associated with the acquisition of resistance to 3'-azido-3'-deoxythymidine (AZT). The results show that mutations in codon 215 of the viral reverse transcriptase can be detected readily by this method in HIV isolates and also directly in peripheral blood from HIV-infected individuals after in vitro amplification of viral sequences with the polymerase chain reaction. The specific recurrence of identical double-nucleotide substitutions in epidemiologically and geographically distant viruses suggests that the restricted amino acid substitutions at this position selected by drug exposure are a critical, rate-limiting step in the acquisition of drug resistance.
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PMID:Characterization of genetic variation and 3'-azido-3'-deoxythymidine- resistance mutations of human immunodeficiency virus by the RNase A mismatch cleavage method. 203 72


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