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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chimeric oligodeoxynucleotides, comprised of internal phosphodiester and terminal methylphosphonodiester sections, possess many beneficial characteristics as antisense effectors. We have investigated the effects of progressive replacement of phosphodiester by methylphosphonodiester linkages on hybrid stability with complementary RNA and DNA. The melting temperatures (Tms) of oligodeoxynucleotide/RNA heteroduplexes were found to decrease dramatically with increasing methylphosphonate substitution. In contrast, a smaller reduction in Tm was observed for comparable DNA heteroduplexes. This disparate reduction in hybrid stability was found with both the G + C-rich human c-myc and A + T-rich human c-Ha-ras sequences used, suggesting that methylphosphonate oligodeoxynucleotide analogues generally hybridize with less affinity to RNA than DNA. RNase H assays were employed to determine if the noted decreases in Tm impaired the ability of chimeric oligodeoxynucleotides to direct the degradation of RNA. Contrary to expectation, increasing methylphosphonate substitution gave rise to increasing rates of RNA degradation for both the c-myc and c-Ha-ras series. The present results suggest that chimeric oligodeoxynucleotide analogues may be of considerable utility as antisense agents in systems where RNase H is thought to make a major contribution to inhibition of gene expression.
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PMID:Enhanced RNase H activity with methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides. 131 29

Several groups have reported the use of antisense oligonucleotides to inhibit c-myc gene expression and study its biological role. However high concentrations of free oligonucleotides were generally needed. To lower their concentration and stabilize the antisense effect against c-myc, oligonucleotides were covalently linked to poly(L-lysine) and administered in ternary complexes formed with heparin (100 micrograms/ml). A sequence specific growth inhibition was observed at concentrations lower than 1 microM, while oligonucleotide-poly(L-lysine) conjugates alone were inefficient. Similar results occurred with other polyanionic compounds. Inhibition of proliferation was correlated to a reduction of c-myc protein and to a transient decrease in c-myc mRNA level. However, implication of RNase H in this process could not be demonstrated.
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PMID:Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene. 170 28

A series of antisense pentadecamers complementary to a variety of target sequences between the cap and AUG initiation codon regions of c-myc mRNA was synthesized and used to treat human promyelocytic leukemia HL-60 cells. The sensitivity of the cap-region sequences to antisense inhibition of c-myc p65 expression was two to three times that of the original initiation codon antisense sequence. The other target sequences downstream of the cap and up to the AUG initiation codon were comparable to the initiation codon sequence, except that the first splice junction was slightly more sensitive. At the primary initiation codon target, a dodecamer was about half as effective as the original pentadecamer, whereas an octadecamer was about twice as effective. The observation of variation in antisense efficacy as a function of target location in c-myc mRNA may represent a combination of the effects of hybrid arrest, RNase H attack, and interference in RNA processing. Alternatively, the most sensitive targets might be those that are the most exposed in the secondary and tertiary structure of c-myc mRNA.
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PMID:Walking along human c-myc mRNA with antisense oligodeoxynucleotides: maximum efficacy at the 5' cap region. 199 45

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.
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PMID:A small nuclear RNA, U5, can transform cells in vitro. 255 90

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA.DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G.C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.
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PMID:Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. 269 70

We developed a modified in situ DNA-RNA hybridization technique and demonstrated c-myc proto-oncogene transcripts in nodular sclerosis type Hodgkin's disease (HD-NS). A hybridization probe was prepared by metabolic labelling of bromodeoxyuridine (BrdU). The hybridized probe was detected with anti-BrdU monoclonal antibody after incubation with ribonuclease H (RNase H), which specifically degrades RNA from DNA-RNA hybrids. In HD-NS, c-myc protooncogene transcripts were demonstrated in the cytoplasm of lacuna cells and a few surrounding lymphoid cells. This modified hybridization method was sensitive and applicable to the study of oncogene expression at a single cell level.
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PMID:In situ hybridization using bromodeoxyuridine labelled DNA probe and RNase H. 839 49

It has been demonstrated that the half-life of c-myc mRNA is modulated in response to physiological agents. The elucidation of the decay process and the identification of the critical steps in the in vivo c-myc mRNA degradation pathway can be approached by following the fate of c-myc mRNA under the influence of such factors. IFN-alpha was the factor used to modulate c-myc mRNA half-life in HeLa 1C5 cells, a stable clone derived from HeLa cells. This cell line carries multiple copies of the c-myc gene, under the control of the dexamethasone inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Exposure of HeLa 1C5 cells to IFN-alpha resulted in a further 2-fold increase over the dexamethasone-induced c-myc mRNA. However, the c-myc mRNA in IFN-alpha treated cells was less stable than that in the control cells. RNase H mapping of the 3' untranslated region of c-myc mRNA revealed, in addition to the full length mRNA, three smaller fragments. These fragments were proven to be truncated, non-adenylated c-myc mRNA species generated in vivo. Exposure of HeLa 1C5 cells to Interferon-alpha before induction with dexamethasone resulted in the enhanced presence of these intermediates. RNase H analysis of c-myc mRNA after actinomycin D chase revealed that deadenylation led to the formation of a relatively more stable oligoadenylated c-myc mRNA population which did not appear to be precursor to the truncated intermediates. The detection of truncated 3' end c-myc mRNA adenylated fragments as well, implies that the c-myc mRNA degradation process may follow an alternative pathway possibly involving endonucleolytic cleavage.
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PMID:In vivo generation of 3' and 5' truncated species in the process of c-myc mRNA decay. 901 68

Expression of messenger RNA (mRNA) in both embryonic and adult cells may be profoundly influenced by untranslated sequences in the 3'-end. Elements in the 3'-untranslated regions (UTRs) of messengers are known to influence messenger stability, polyadenylation, and translation. We have examined the effects of the 3'-UTR of Xenopus laevis c-mycI (either alone or in combination with the 5'-first exon) on the expression of a chloramphenicol acetyltransferase (CAT) reporter in Xenopus embryos. The Xenopus c-mycI 3'-UTR enhanced messenger translation independent of the 5'-UTR. RNase H analysis indicated that the Xenopus c-mycI 3'-UTR can promote the cytoplasmic polyadenylation of CAT mRNA in embryos. The result suggests that the post-fertilization enhancement of translation caused by the c-mycI 3'-UTR may be a consequence of cytoplasmic polyadenylation. A uridine (U)-rich sequence in the Xenopus c-mycI 3'-UTR that may be responsible for polyadenylation is similar to an element that destabilizes mammalian c-myc transcripts. We discuss the possibility that U-rich sequences may play a dual role by destabilizing growth-related transcripts in adult cells and stimulating their polyadenylation during development, and we propose that a switch in the role of such sequences in adult cells could lead to stabilization of these messengers, increased translation, and abnormal growth control.
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PMID:Stimulation of translation and cytoplasmic polyadenylation by the Xenopus c-mycI 3'-untranslated region. 940

During the course of a study aimed at improving antisense oligodeoxynucleotide-mediated ex vivo bone marrow purging of patients suffering from chronic myeloid leukemia (CML), the properties of a number of antisense structures intended to reduce the expression of c-myc, mutant p53, and bcr-abl mRNAs and proteins were examined. The majority of the antisense oligodeoxynucleotides were designed to be capable of directing ribonuclease H (RNase H) cleavage of their target mRNAs. Streptolysin O (SLO) reversible permeabilization was used to deliver the oligodeoxynucleotides into the CML line KYO-1. We found that the efficiency and specificity of antisense oligonucleotide-induced reductions of target protein expression depended on target protein half-life, the oligonucleotide structure, and the specific sequence within the target mRNA. Transient reductions of c-myc mRNA and protein were achieved with a chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to the initiation codon, but cell proliferation was unaffected. In contrast, a chimeric oligodeoxynucleotide of similar structure targeted to an alternative site in the coding region of c-myc mRNA reduced target mRNA and protein levels for over 24 hours and halted cell proliferation. Chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to a point mutation in KYO-1 p53 mRNA efficiently reduced target mRNA expression, but only small, transient reductions in p53 protein expression were observed. However, a chimeric methylphosphonate-phosphorothioate oligodeoxynucleotide targeted to the same site reduced p53 protein to 30% of control levels over a 48-hour period. BCR-ABL protein expression was unaffected by chimeric oligodeoxynucleotides targeted to the breakpoint in bcr-abl mRNA, even when mRNA levels at early times were substantially reduced.
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PMID:The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses. 974 66

A 28-mer morpholino oligonucleotide analog was designed to hybridize to 8 bases of intron 1 and extend 2 bases beyond the translation initiation codon in exon 2 of the unspliced c-myc RNA transcript. Delivery of this compound into human chronic myeloid leukemia KYO1 cells, by streptolysin O permeabilization, resulted in almost total ablation of the 65 kDa c-MYC protein expression for at least 24 hours after treatment. An unexpected band with SDS-PAGE electrophoretic mobility indicating a protein of about 47 kDa was apparent on the 24-hour western blots that were developed using antibodies that recognize MYC protein C terminal epitopes. No inhibition of the approximately 2400 nt c-myc mRNA expression was observed by northern hybridization, a result of the inability of morpholino analogs to direct the activity of ribonuclease H. In fact, high molecular weight c-myc RNA species were found to have accumulated in antisense-treated KYO1 cells. Control sense and scrambled antisense morpholino analogs did not inhibit MYC protein expression or induce the appearance of the anomalous RNA and protein bands. Molecular analyses by RT-PCR and sequencing revealed that the morpholino antisense effector had (1) inhibited splicing of the c-myc pre-mRNA, (2) induced missplicing of the pre-mRNA, and (3) inhibited translation of normal spliced c-myc mRNA. Identical results were obtained with acute promyelocytic leukemia, acute lymphoblastic leukemia, and histiocytic lymphoma cell lines.
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PMID:Antisense morpholino oligonucleotide analog induces missplicing of C-myc mRNA. 1035 27


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