Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antisense oligonucleotide (AS-ODN) inhibition of angiotensin receptors (AT1-R) offers a potentially novel therapeutic approach for hypertension, left ventricular hypertrophy and other aspects of cardiovascular disease. To clarify questions concerning cellular uptake and retention of these oligos, we quantified the trafficking and stability of phosphorothioated modified AS-ODN to AT1 receptor mRNA in adrenal cells, using visual and chromatographic analysis. The AS-ODN to AT1 receptor mRNA was effective in significantly inhibiting AT1 receptor binding in a dose dependent manner. FITC-labeled ODNs were used to determine the cellular uptake in bovine adrena cortex cells; using confocal microscopy, rapid cellular uptake of 15-mer ODNs was observed. Uptake is initially rapid (30 min to 4 h) followed by a slower uptake process 24 h and after. The cellular accumulation of ODN involves a dynamic balance between influx and efflux processes. Efflux of FITC-ODN had a f1/2 = 4.6 days. Uptake was time and dose dependent. No obvious degradation of intracellular ODNs occurred as shown by intact peaks for 15-mer ODN on thin layer chromatography. The results suggest that the AS-ODN to AT1 receptor mRNA was resistant to cellular nucleases. The FITC-ODN accumulated mainly in the nucleus and remained there intact for up to 3 days. No significant change in target mRNA was observed by quantitative RT-PCR. Therefore the antisense inhibition mechanism of this ODN does not appear to stimulate RNase H or block transcription. Since the ODN accesses the nucleus, the results imply that the ODN inhibits specific mRNA transport into the cytoplasm. The data show that AS-ODN, for inhibition of AT1 receptors, is rapidly taken up and stable in cells and produces specific inhibition of AT1 receptors.
Neurochem Int 1997 Sep
PMID:Uptake and efflux of intact antisense phosphorothioate deoxyoligonucleotide directed against angiotensin receptors in bovine adrenal cells. 924 81

Petunia vein-clearing virus (PVCV) is a plant pararetrovirus that has some features of retrotransposons. It encapsidates dsDNA and has isometric particles and inclusion bodies similar to those of caulimoviruses. The PVCV genome of 7205 bp has two large ORFs in the transcribed strand and a methionine tRNA primer-binding site in its 663-bp intergenic region. The N-terminal position of the large protein (126 kDa) encoded by ORF I has similarity to the movement protein of caulimoviruses. Toward the C-terminus of this same polyprotein are the two distinctive sequence elements [HHCC and DD(35)E] of the integrase function of retroviruses and retrotransposons. ORF II of PVCV encodes a protein of 125 kDa with domains for an RNA-binding element, common to the gag gene of retroelements, followed by consensus sequences for an acid protease, reverse transcriptase, and ribonuclease H. Hence, the gag equivalent (capsid protein) and pol gene of PVCV are part of the same polyprotein. Phylogenetic comparison of the reverse transcriptase of PVCV with that of various other retroelements grouped PVCV between caulimoviruses and the Ty3/gypsy retrotransposons, suggesting that PVCV is a divergent member of the caulimoviruses.
Virology 1997 Sep 15
PMID:Petunia vein-clearing virus: a plant pararetrovirus with the core sequences for an integrase function. 929 26

Conjugates of mitomycin C (MC) and 15-mer oligodeoxyribonucleotides (ODNs) were synthesized in which the 7-amino group of MC was tethered by either a (-CH2-)6 or a (-CH2-)12 linker to the 5'-terminal phosphate of the ODNs. The conjugates were shown to be cross-linked selectively to complementary 18-mer oligoribonucleotides (ORNs). The cross-linking was dependent on reductive activation of the MC moiety of the conjugates by NADPH-cytochrome c reductase/NADPH. The cross-linked ODN-ORN hybrid duplexes were characterized as such by degeneration by RNase H. Cross-linking efficiencies of the conjugates were 50 and 25% in the case of the (-CH2-)12 tether and the (-CH2-)6 tether respectively The results demonstrate the feasibility of sequence-targeted alkylation of RNA by MC via antisense recognition.
Anticancer Drug Des 1997 Sep
PMID:Antisense sequence-directed cross-linking of RNA oligonucleotides by mitomycin. 931 56

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.
EMBO J 1997 Sep 01
PMID:Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex. 931 86

The catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1). The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed in Escherichia coli, and purified by affinity chromatography. This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10. The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity. Inhibitor sensitivity to ddTTP and 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes. However, the two enzymes differed dramatically in their sensitivity to several inhibitors. While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50 ranged from 0.16 to 8.2 microM, depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50 > 200 microM). The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors. The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1.
Virology 1997 Sep 29
PMID:Characterization of the reverse transcriptase of a human immunodeficiency virus type 1 group O isolate. 932 44

Oligodesoxyribonucleotide-directed cleavage of protein-deficient Thermus thermophilus derivatives of the 30S ribosomal subunits with RNase H is described. A homogeneous RNP fragment has been isolated as a result of the cleavage and subsequent purification in the sucrose gradient. It corresponds to the central and 5' domains of the 30S ribosomal subunit. The high compactness of the fragment in solution suggests that it can be considered as a 'beheaded' derivative of the 30S ribosomal subunit. The absence of a reconstitution stage in isolation of the 22S RNP fragment provides for its preparation in large amounts.
Biochimie 1997 Sep
PMID:Preparation of a 'beheaded' derivative of the 30S ribosomal subunit. 945 54

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.
J Virol 1998 Sep
PMID:Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus. 969 91

Spumaviruses, or foamy viruses, express a pol-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.
J Virol 1998 Sep
PMID:Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease. 969 69

During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3'-DNA-RNA-5' junction is formed involving the 3'-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 A resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.
Biochemistry 1998 Sep 01
PMID:Crystal structure of an eight-base pair duplex containing the 3'-DNA-RNA-5' junction formed during initiation of minus-strand synthesis of HIV replication. 972 10

Homodimeric EIAV p51/51 and heterodimeric EIAV p66/51 reverse transcriptase were purified in order to compare the different modes of DNA synthesis supported by the enzymes. Analysis of the dimerization behavior of the EIAV enzymes indicates that the dimer stability of EIAV reverse transcriptase enzymes is higher than that of their HIV-1 reverse transcriptase counterparts. EIAV p51/51 polymerizes DNA distributively whereas DNA synthesis by EIAV p66/51 is processive. Steady-state and pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation were performed with both enzymes to determine the reasons for the different polymerization behavior. Equilibrium fluorescence titrations demonstrated that the Kd values of EIAV p51/51 for binding of DNA/DNA and DNA/RNA substrates are increased 10-fold and 28-fold, respectively, as compared to EIAV p66/51. Stopped-flow measurements with DNA/DNA show that the increase in the Kd is in part due to a 17. 4-fold higher dissociation rate constant (k-1) for EIAV p51/51. Additionally, with EIAV p51/51, kdiss is increased 7-fold for DNA/DNA and 14-fold for DNA/RNA primer/template substrates, respectively. The lack of the RNase H domain in EIAV p51/51 leads to differences in the pre-steady-state kinetics of nucleotide incorporation on DNA/DNA and DNA/RNA templates. The burst of both enzymes is composed of two phases for both substrates, and the values for the corresponding pre-steady-state burst rates, kpol1 and kpol2, are similar for both enzymes, implying the formation of identical polymerase active sites. However, the amplitudes of the two phases differ with DNA/DNA templates, indicating a different distribution between two states varying greatly in their kinetic competence.
Biochemistry 1998 Sep 01
PMID:Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase. 972 26


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