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At least 811 records · Page 45Linked to original sources
PubMed2003

A new type of RNase T2 ribonuclease in two Basidiomycetes fungi, Lentinus edodes and Irpex lacteus.

Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.

Amino Acid Sequence↗
PubMed2005

A convenient and sensitive fluorescence resonance energy transfer assay for RNase L and 2',5' oligoadenylates.

Interferon action against viruses is mediated in part through a ribonucleic acid (RNA) decay pathway known as the 2-5A system. Unusual 5'-triphosphorylated, 2',5'-linked oligoadenylates (2-5A) are produced in mammalian cells by interferon-inducible 2-5A synthetases (OAS) in response to viral double-stranded RNA. 2-5A activates a uniquely regulated endoribonuclease, RNase L, resulting in the cleavage of single-stranded viral and cellular RNAs, thus suppressing viral replication. In addition, RNase L was recently identified as a strong candidate for the hereditary prostate cancer 1 susceptibility allele. RNase L is ubiquitously expressed at basal levels in a wide range of mammalian cell types. Conventional RNase L assays, which can be inconvenient and cumbersome, typically involve cleavage of radioactively labeled RNA species or of endogenous ribosomal RNA. Here we describe a convenient, rapid, nonradioactive, and relatively inexpensive fluorescence resonance energy transfer (FRET) that may be used to accurately measure levels of either 2-5A or RNase L activity with a high degree of specificity and sensitivity. The RNA probe used in the FRET assay was designed based on a region of respiratory syncytial genomic RNA. We demonstrate the utility of our FRET assay with several novel biostable analogs of 2-5A.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

Preparation of recombinant RNase single-chain antibody fusion proteins.

This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni2+-NTA agarose), results in the final purification of the RNase fusion protein.

Animals↗
PubMed1998

RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag).

The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA. RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally. Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity. During this period an mRNA fragment, lacking an intact 5' end, accumulates. This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway. During this lag, the levels of bulk protein are maintained. Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization. Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

Down-Regulation↗
PubMed2002

Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format.

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.

Bacteriological Techniques↗
PubMed1996

Cycling probe technology with RNase H attached to an oligonucleotide.

A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits. Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties. Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme. The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe. These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.

Bacterial Proteins↗
PubMed1987

[Clinical significance of serum ribonuclease (RNase) assay in acute leukemia].

The clinical significance of serum ribonuclease (RNase) assay in acute leukemia was studied. Serum RNases were assayed by the method of Akagi et al. with slight modifications in the serum samples obtained from 50 cases of healthy subjects, 55 cases of acute leukemia before therapy, 18 chronic myelocytic leukemia before therapy, 13 chronic myelocytic leukemia under treatment and 20 reactive leukocytosis. The ratio of acid RNase to alkaline RNase activities (Ac/Al ratio) was statistically increased in acute promyelocytic leukemia, acute myeloblastic leukemia [M2], acute myelocytic leukemia and erythroleukemia (leukemic stage) compared with those in healthy subjects (P less than 0.001). All cases of acute promyelocytic leukemia and most of the acute myeloblastic leukemia [M2], acute myelomonocytic leukemia and erythroleukemia cases had an Ac/Al ratio of above 1.0. In remission of acute leukemia, it is noteworthy that acid and alkaline activities showed no substantial difference from those of healthy subjects. While, on relapse of acute leukemia cases, showing Ac/Al ratio above 1.0 in pretreatment state, Ac/Al ratio increased to above 1.0. Thus, the assay of serum RNases and the calculation of Ac/Al ratio might be an additional method for diagnosing acute leukemia and for assessing their remission and recurrence in some type of acute leukemia.

Acute Disease↗
PubMed2000

An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression.

RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.

5' Untranslated Regions↗
PubMed2002

[Mechanism of heavy metal ions on RNase activity from bovine pancreas].

This paper studied mechanism of Ce3+, Cd2+, Pb2+ on RNase activity from bovine pancreas. The results showed that the activity of RNase was enhanced under the treatment by Ce3+, Cd2+, Pb2+ at lower concentration (10-60 or 10-30 mumol.L-1), but was inhibited by Ce3+, Cd2+, Pb2+ at higher concentration (40 or 70 mumol.L-1 above), and the inhibition was in the order as Pb2+ > Cd2+ > Ce3+. The equilibrium dialysis demonstrates that RNase may have one Ca(2+)-binding site. The fluorescence titration showed that one molecule of RNase has one binding site for Ce3+, the association constant k for its low-affinity Ce(3+)-binding site is 1.22 x 10(8) L.mol-1. However, it can bind three Cd2+ or Pb2+ and the association causing constant k for its low-affinity Cd2+ or Pb(2+)-binding site is 1.8 x 10(8) L.mol-1, 2.01 x 10(8) L.mol-1, respectively, and caused the conformational changes of RNase.

Animals↗
PubMed1992

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Amino Acid Sequence↗
PubMed1992

RNA abundance measured by a lysate RNase protection assay.

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.

Genetic Techniques↗
PubMed1990

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.

Base Sequence↗
PubMed1991

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small.

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.

Base Sequence↗
PubMed1991

Microwave exposure alters the expression of 2-5A-dependent RNase.

The effects of 2.45-GHz continuous-wave microwaves (SAR = 130 mW/g) on the expression of the interferon-regulated enzymes 2'-5'-oligoadenylate (2-5A) synthetase(s) and 2-5A-dependent endoribonuclease (RNase L) were studied in murine L929 cells. Cells growing as monolayers were removed from the substratum and placed in suspension culture for a 4-h sham or microwave exposure. The cells were returned to monolayer growth for 18 h, and then harvested and assayed to determine the amount of RNase L protein (via [32P]2-5A binding) and the specific activities of RNase L and 2-5A synthetase. Binding of radioactive 2-5A to RNase L for sham- and microwave-exposed samples was 14.5 and 36.4% above control, respectively (the microwave-exposed bound 19.0% more probe than the sham-exposed). The increases in 2-5A binding were accompanied by corresponding elevations of RNase L specific activity. In contrast, sham or microwave irradiation produced no alterations in 2-5A synthetase specific activity. No detectable differences were noted in the postexposure cell viability, plating efficiency, or proliferation rate. Also, there were no detectable differences in cell viability or plating efficiency between controls and cultures irradiated for 2 h when the temperature was simultaneously increased to above normal physiological limits (39 to 45 degrees C). The SAR (130 mW/g) and the power density (95 mW/cm2) used for the greater part of this study were nearly 20 times higher than the ANSI limit of 8 mW/g and 5 mW/cm2 for any 1 g of exposed human tissue.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease.

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.

Avian Leukosis Virus↗
PubMed1989

[Changes in serum RNase activities in chronic phase and acute crisis of chronic myelocytic leukemia].

We investigated serum acid and alkaline RNase activities in 16 cases of acute crisis of chronic myelocytic leukemia (CML), 21 cases of untreated CML, and 13 cases of treated CML, to clarify clinical significance of the determination of RNase activities in acute crisis of CML. We obtained results as follows; the ratio of acid to alkaline Rnase activities (Ac/Al ratio) of chronic phase of CML was 1.64 +/- 0.47 (mean +/- SD) in the untreated cases, and was 1.32 +/- 0.16 in the treated cases. The Ac/Al ratio always indicated over 1.0 in the chronic phase of CML without any relationship to treatments. On the other hand, in the cases of acute crisis, the Ac/Al ratio was significantly lowered (0.94 +/- 0.22) as compared to the chronic phase of CML (p less than 0.001), and was similar to that of acute leukemia. The acid and alkaline RNase activities of the blast from the patients with acute crisis of CML showed remarkably lower value than those of leukemic cells from patients with chronic phase of CML. Therefore, it was Suggested that the return to normal range of Ac/Al ratio in acute crisis of CML depended on marked decrease of RNase activities of blasts. Thus, serial determinations of the enzyme activities are considered to be one of useful tools for prediction of acute crisis of CML.

Adult↗
PubMed1988

Transfer RNA is a substrate for RNase D in vivo.

RNase D is a 3'-exonuclease whose in vitro specificity has suggested a role in tRNA processing. However, since mutant Escherichia coli strains devoid of RNase D display a normal phenotype, it has not been possible to ascertain the enzyme's function or even to determine which RNA is its substrate in vivo. Here we show that transformation of strains devoid of tRNA nucleotidyltransferase with a multicopy plasmid carrying the rnd+ gene leads to extremely slow growth due to elevated levels of RNase D activity. Analysis of such a slow-growing strain revealed that less tRNA is present in the cell and that the tRNA that could be recovered is substantially damaged. These studies demonstrate that RNase D can act at the 3' terminus of tRNA in vivo, and they support the conclusion that RNase D participates in tRNA metabolism.

Endoribonucleases↗
PubMed1979

Identification of two different RNase H activities associated with yeast RNA polymerase A.

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.

Chromatin↗