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)

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.
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PMID:Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease. 156 48

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.
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PMID:Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell. 871 Mar 70

Oligonucleotide N3'-->P5'phosphoramidates are a new and promising class of antisense agents. Here we report biological properties of phosphoramidate oligonucleotides targeted against the human T cell leukemia virus type-I Tax protein, the major transcriptional transactivator of this human retrovirus. Isosequential phosphorothioate oligodeoxynucleotides and uniformly modified and chimeric phosphoramidate oligodeoxynucleotides containing six central phosphodiester linkages are all quite stable in cell nuclei. The uniformly modified anti-tax phosphoramidate oligodeoxynucleotide does not activate nuclear RNase H, as was shown by RNase protection assay. In contrast, the chimeric phosphoramidate-phosphodiester oligodeoxynucleotide is an efficient activator of RNase H. The presence of one or two mismatched nucleotides in the phosphodiester portion of oligonucleotides affected this activation only negligibly. When introduced into tax-transformed fibroblasts ex vivo, only the uniformly modified anti-tax phosphoramidate oligodeoxynucleotide caused a sequence-dependent reduction in the Tax protein level. Neither the chimeric phosphoramidate nor the phosphorothioate oligodeoxynucleotides significantly reduced tax expression under similar experimental conditions.
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PMID:RNase H-independent antisense activity of oligonucleotide N3 '--> P5 ' phosphoramidates. 901 28

Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC-II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.
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PMID:Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy. 923 82

Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.
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PMID:Mouse hypoxia-inducible factor-1alpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter. 955 7

The transformation-associated region of herpesvirus saimiri strains is variable, whereas other parts of the virus genome are highly conserved. However, we observed considerable interstrain sequence divergence of the early viral regulatory orf50 gene, which encodes the R transactivator, a homolog of Epstein-Barr virus BRLF1. The orf50 gene of strain C488 was transcribed at low abundance during lytic infection, whereas antisense transcripts were simultaneously expressed at high levels. A spliced variant, orf50a, was detectable by RT-PCR and RNase protection assays in stimulated C488-transformed, nonpermissive human T cells. In contrast to strain A11, the short, unspliced orf50b form of C488 displayed complete transactivation capability on the orf6 and orf57 promoters. In summary, there are unexpected structural and functional differences between the orf50 genes of herpesvirus saimiri strains, which differ in their capability to transform human T lymphocytes.
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PMID:Distinct transcriptional and functional properties of the R transactivator gene orf50 of the transforming herpesvirus saimiri strain C488. 1068 39

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.
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PMID:AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. 1091 88

Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.
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PMID:Early shutoff of host protein synthesis in cells infected with herpes simplex viruses. 1208 25

The 3' untranslated region (UTR) of rTSalpha RNA is complementary (i.e., antisense) to human thymidylate synthase (TS) RNA. When HEp2 cells (human epidermoid carcinoma) progressed from late-log to plateau phase growth, ribonuclease protection assay (RPA) revealed an inverse correlation between the levels of rTSalpha RNA and TS mRNA, suggesting a possible effect of rTSalpha RNA on TS mRNA levels. HEp2 cells expressing a Tet-On transactivator were transiently co-transfected with pHook-1 and a construct containing rTSalpha (protein and antisense RNA), rTSalphaDelta3' (rTSalpha protein only), rTSalpha-3' (antisense RNA-luciferase) or luciferase. Transfected cells were selected and evaluated for the effects of induced transgene expression on TS mRNA. Induced expression of transfected rTSalpha or rTSalpha-3', but not rTSalphaDelta3' or luciferase, resulted in decreased TS mRNA levels as measured by RPA. These results demonstrated that the antisense region of rTSalpha RNA is necessary and sufficient for this down-regulation of TS mRNA. RPA for TS mRNA also showed the enhanced appearance of two partial-length protected fragments in rTSalpha or rTSalpha-3' transfected cells. RPA stringency evaluations and primer extension assays indicated that TS mRNA is cleaved in vivo in a site-specific manner. These data demonstrate that rTS gene expression likely plays a role in down-regulating TS through a natural RNA-based antisense mechanism.
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PMID:Natural antisense (rTSalpha) RNA induces site-specific cleavage of thymidylate synthase mRNA. 1208 60